Nitroreductase enzymes

ABSTRACT

Nitroreductases, and genes encoding the same, are provided that demonstrate preferential catalytic conversion of the alkylating agent CB1954 into its highly cytotoxic 4-hydroxylamine (4HX) derivative, this derivative demonstrating anticarcinoma properties. Accordingly, the catalytic activity of the nitroreductase enzymes of the present invention may be employed to achieve catalysis of CB1954 into its cytotoxic derivative in a site-directed manner, such as by Directed-Enzyme Prodrug Therapy (DEPT).

[0001] The present invention relates to polypeptides and proteins havingnitroreductase activity, to DNA and genes encoding these nitroreductasesand to methods of obtaining such enzymes, DNA and genes.

[0002] A number of cancer therapies are based upon or exploit theconversion of a non-toxic prodrug into a toxic derivative.

[0003] One example concerns the monofunctional alkylating agent CB1954,which exhibits extreme toxicity towards the Walker 256 rat carcinoma asa result of the presence of a DT-diaphorase enzyme (DTD) which reducesthe 4-nitro group of CB1954 to give a highly cytotoxic 4-hydroxylamine(4HX) derivative. CB1954 does not have the same effect on humancarcinomas because human cells lack this enzyme but would be effectiveagainst human tumours if an enzyme such as DTD were externally supplied,e.g. in a Directed-Enzyme Prodrug Therapy (DEPT). The rat DTD, however,has a relatively poor specific activity for CB1954. The E. coli Bnitroreductase enzyme (NfnB) was isolated as a more effectivealternative and is the subject of EP-A-0540263. It exhibits a higherspecific activity for CB1954, compared with the rat enzyme and is,therefore, currently the preferred enzyme in anti-cancer DEPTstrategies.

[0004] Whilst the known E. coli enzyme receives widespread attentionfrom cancer biologists seeking to develop gene based DEPT strategies, ithas a number of drawbacks. These mostly relate to its activity againstthe preferred prodrug, CB1954—it has a relatively high K_(m) and lowK_(cat), and converts CB1954 into equimolar amounts of a relativelyinnocuous 2-hydroxylamino derivative (2HX) in addition to the highlycytotoxic 4-hydroxylamino species (4HX).

[0005] In relation to this specific prodrug, it is hence desired toprovide an alternative to the known E. coli enzyme.

[0006] Additionally, and more generally, analogues of CB1954 andprodrugs other than CB1954 are known and further such precursors ofpotential toxic agents may become the focus of future therapies. Inrelation to all of these it is desired to provide further enzymescapable of use in converting prodrugs into drugs, e.g. for clinicaluses.

[0007] It is an object of the present invention to providenitroreductase enzymes, in particular nitroreductase enzymes forconverting CB1954 and analogues thereof into drugs. It is a furtherobject of the present invention to provide DNA and genes encodingnitroreductases, which DNA and genes in particular are incorporated intopharmaceutical compositions for prodrug therapies.

[0008] The present invention is based upon the discovery, purification,gene sequencing and/or expression of nitroreductases in bacteria andother microorganisms with hitherto unknown properties in convertingprodrugs such as CB1954 into toxic derivatives. These nitroreductasesposses properties which alone or in combination offer potentialimprovements compared with the known enzymes in this technology. Thenitroreductases of the invention may be divided into different familiesbased upon such characteristics as activity, product spectrum and/oramino acid sequence, and each given nitroreductase may fall into morethan one of these families.

[0009] The present invention provides, in a first aspect, anitroreductase enzyme, characterised in that it preferentially reducesCB1954 to a product that is a cytotoxic 4-hydroxylamine (4HX)derivative.

[0010] The enzymes of this aspect of the present invention confer theadvantage that the product they generate from CB1954 contains a greaterproportion of the cytotoxic 4HX derivative then the non-cytotoxic2-hydroxylamino derivative. In preferred embodiments of the invention,the product is substantially entirely the cytotoxic derivative. Theenzymes may hence be more efficient that those of the art as the enzymesof the invention produce more cytotoxic product for a given amount ofpro-drug.

[0011] The present invention further provides, in a second aspect, anitroreductase enzyme, characterised in that it reduces a prodrug to atoxic derivative with a K_(m) of less 700 micromolar, wherein theprodrug is selected from CB1954 and analogues thereof or otherbioreductive drugs (Denny et al, B. J. Cancer, 1996, 74, pp S32-S38).The enzymes of the second aspect of the invention offer an advantageover the known E. coli derived enzyme in that they have a lower K_(m)(K_(m) of E. coli NfnB for CB1954 is around 862 micromolar) and thushave a higher affinity for substrate. Twenty nitrogen mustard analoguesof CB1954 are described by Friedlos et al (J Med Chem, 1997, 40,1270-1275).

[0012] More preferably, the K_(m) of the enzymes of the second aspect ofthe invention is less than 300 micromolar.

[0013] In a third aspect, the present invention provides anitroreductase enzyme characterised in that it reduces a prodrug to atoxic derivative with a K_(cat) of at least 8, wherein the prodrug isselected from CB1954 and analogues thereof.

[0014] The enzymes of this aspect of the invention offer an improvementover that of the art, specifically the E. coli enzyme, in that they havean improved K_(cat)—i.e a higher value than for E. coli NfnB indicatinga higher turnover of substrate by the enzyme. In preferred embodimentsof this aspect of the invention, the K_(cat) of the enzymes is at least10.

[0015] In a fourth aspect of the invention, there is provided anitroreductase enzyme characterised in that it reduces CB1954 to a toxicderivative, it reduces SN23862 to a toxic derivative, it can use NADHand/or NADPH as electron donor and in that it shares no more than 50%sequence identity with the E. coli NfnB sequence. Preferably, thesequence identity is about 25% or less, this sequence identity beingmeasured using the MEGALIGN (registered trade mark) software.

[0016] It has already been discussed how the known E. colinitroreductase is well characterised and is fully sequenced. Thenitroreductases of the fourth aspect thus represent a class of enzymeshaving nitroreductase activity, or being nitroreductase-like, whichnevertheless are so different in amino acid sequence from the E. colienzyme as to represent a separate family of nitroreductases.

[0017] This aspect of the invention thus advantageously provides afurther class of nitroreductase enzymes for use e.g. in prodrugtherapies.

[0018] The invention still further provides, in a fifth aspect, anitroreductase enzyme characterised in that it reduces CB1954 or ananalogue thereof to a toxic derivative, in that it shares at least 50%sequence identity with the rat DTD sequence and in that it does notcontain a domain that is the same as or corresponds to amino acids 51 to82 of the rat DTD sequence.

[0019] Sequence identity is suitably measured in the same way asdescribed above in relation to the fourth aspect.

[0020] To determine whether a given nitroreductase contains a domainthat is the same as or corresponds to amino acids 51 to 82 of the ratDTD sequence, the amino acid sequence of the given nitroreductase and ofthe rat DTD sequence are aligned using a conventional sequence alignmentprogram, such as MEGALIGN (registered trade mark) made by DNASTAR, Inc.

[0021] If the alignment program indicates that there are no amino acidsin the given sequence that, following the algorhythm of the program, areheld to correspond to those at positions 51-82 of the rat DTD sequencethen it is concluded that the rat domain is lacking from the givensequence.

[0022] This aspect of the invention thus provides a further class ofnitroreductase enzymes for conversion e.g. of prodrugs into drugs. Anitroreductase in this class may also be obtained by deleting amino acidresidues that correspond to residues 51-82 of the rat DTD from a knownmammalian enzyme.

[0023] The nitroreductases of the invention may also be NADPH dependant.This property further distinguishes some enzymes of the invention fromthe known E. coli enzyme and the rat DTD.

[0024] It has been found that enzymes having one or more of theproperties described may be obtained from bacteria of the familyBacillus, in particular a Bacillus selected from B. amyloliquefaciens,B. subtilis, B. pumilis, B. lautus, B. thermoflavus, B. licheniformisand B. alkophilus. This finding is of surprise in that at least threenitroreductase enzymes have been found in some species, in particular B.subtilis, B. lautus and B. pumilis, and as nitroreductases having theadvantageous properties of the invention have not hitherto beenidentified in these bacteria, the currently used nitroreductase beingobtained from E. coli.

[0025] In specific embodiments of the invention described in more detailbelow, a nitroreductase has a sequence selected from SEQ ID Nos 2, 4, 6,8, 10, 12, 14, 16, 17, 18, 19, 20, 21, 23, 25, 27 and 29.

[0026] It has further been found that nitroreductases according to theinvention may fall into more than one aspects of the invention. It ishence preferred that a nitroreductase of the invention possesses theproperties of at least two aspects of the invention, and more preferablyat least three aspects of the invention.

[0027] A specific embodiment of the invention is a nitroreductase of SEQID NO: 2 obtained from B. amyloliquefaciens this enzyme converts CB1954into substantially only the cytotoxic derivative, hence falling into thefirst aspect of the invention, but also has a K_(m) that is improvedcompared to the E. coli enzyme, hence falling also into the secondaspect of the invention.

[0028] A further specific embodiment of the invention is anitroreductase from B. subtilis, SEQ ID NO: 9. This enzyme has a betterK_(cat) than the E. coli enzyme, its K_(cat) being about 15 comparedwith about 6 for the E. coli enzyme, and hence falls into the thirdaspect of the invention. Additionally, this enzyme falls into the fourthaspect of the invention in that it reduces both CB1954 and SN23862 butshares less than 30% sequence identity with the E. coli sequence.Another B. subtilis enzyme, SEQ ID NO: 11 is similarly in both the thirdand fourth aspects of the invention, having a K_(cat) of about 15.

[0029] From the examples set out below it will be apparent how thefurther specific embodiments of the invention fall into at least two andeven three aspects of the invention.

[0030] The enzymes of the invention are of use in enzyme directedprodrug therapy. Accordingly, it is preferred that they are provided inpurified form.

[0031] A sixth aspect of the invention provides a pharmaceuticalcomposition comprising a nitroreductase enzyme according to any of thefirst to fifth aspects of the invention in combination with apharmaceutically acceptable carrier.

[0032] As mentioned above, the nitroreductase of the invention are ofuse in therapies such as directed-enzyme prodrug therapies. In thesetherapies, it is required to deliver the nitroreductase to the targetsite. This delivery can be achieved by delivering the enzyme itself orby delivering a DNA or gene coding for the enzyme.

[0033] In an example of the enzyme of the invention in use, apharmaceutical composition is designed for a directed-enzyme prodrugtherapy, and comprises a pharmaceutically acceptable carrier and acompound for converting a prodrug into a drug, wherein a compound iscomposed of at least a nitroreductase according to any of the first tofifth aspects of the invention conjugated to a targeting moiety.

[0034] The targeting moiety can suitably comprise an antibody specificfor a target cell. Alternatively, the targeting moiety is a moietypreferentially accumulated by or taken up by a target cell.

[0035] A further example of delivery of the enzyme of the invention isachieved in a gene therapy-based approach for targeting cancer cells, asdescribed in WO 95/12678. As described by Knox R. J. et al, the basis ofthis further prodrug therapy is delivery of a drug susceptibility geneinto target, usually tumour or cancer, cells. The gene encodes anitroreductase that catalyses the conversion of a prodrug into acytotoxic derivative. The nitroreductase itself is not toxic andcytotoxicity used to treat the tumour cells arises after administrationof a prodrug which is converted into the cytotoxic form. A bystandereffect may be observed as cytotoxic drug may diffuse into neighbouringcells.

[0036] Thus, in this gene-based therapy, the nitroreductase is expressedinside a cell, in contrast to other delivery systems in which, forexample, the enzyme itself is delivered accompanied by a targetingmoiety.

[0037] Targeting of gene-based therapies may be achieved by providing avirus or liposome with altered surface components so that the deliveryvehicle is recognised by target cells. Typically, transcriptionalelements are chosen so that the gene coding for the nitroreductaseenzyme will be expressed in the target cells, and preferablysubstantially only in the target cells. A number of viral-based vectorsare suitable for this delivery. Retro-viral based vectors typicallyinfect replicating cells. Adenoviral vectors and lentiviral-vectors arealso believed to be suitable.

[0038] This delivery technology has been demonstrated by Bridgewater etal (Eur J Cancer 31a, 236-2370,1995). A recombinant retrovirus encodinga nitroreductase was used to infect mammalian cells, it being observedthat infected cells expressing the nitroreductase were killed byapplication of CB1954.

[0039] Accordingly, a further aspect of the invention provides the useof a DNA sequence coding for a nitroreductase of the invention inmanufacture of a medicament for prodrug therapy.

[0040] The medicament may take the form of a viral vector, comprising aDNA encoding the nitroreductase of the invention operatively coupled toa promoter for expression of the DNA. The medicament may take the formof a mini-gene comprising a DNA operatively linked to a promoter forexpression of the DNA, the mini-gene being suitable for inclusion orincorporation into a targeting vehicle such as a microparticle.

[0041] Thus, an embodiment of the invention provides a viral vectorcomprising a nucleotide sequence encoding a nitroreductase according toany of aspects 1 to 5 of the invention, which nitroreductase converts aprodrug into a cytotoxic drug, and also a kit comprising the viralvector and the prodrug, and also a method of treatment of tumours whichcomprises administering an effective amount of the viral vector togetherwith an effective amount of the prodrug.

[0042] The preparation and administration of these viral vectors may besubstantially as described in WO 95/12678, the contents of which isincorporated herein by reference. The present invention relates toproviding nitroreductase enzymes and genes and DNA coding therefore. Theuses of those enzymes and genes may be as set out in WO 95/12678.

[0043] A nitroreductase can also be delivered by putting a gene of theinvention into a bacteria that selectively colonises tumours, such as aclostridial (Lemmon et al, Gene Therapy, 1997, 4, 791-796) or Salmonellaspecies.

[0044] A further aspect of the invention provides an isolated DNAencoding a nitroreductase according to any of the first to fifth aspectsof the invention. The DNAs of this further aspect of the invention, andalso the DNAs incorporated into vectors of the invention, preferablycomprise a sequence which is selected from SEQ ID NOs: 1, 3, 5, 7, 9,11, 13, 15, 22, 24, 26 or 28, together with fragments, derivatives andanalogs thereof retaining nitroreductase activity according to one ofthe first to fifth aspects of the invention. The fragments, derivativesand analogs are suitably selected from sequences which retain at least70% identity with the specific embodiments of the invention, orpreferably at least 90% identity and most preferably at least 95%identity.

[0045] The enzymes of the invention can also be obtained by purificationfrom cell extracts and may also be obtained by recombinant expression ofDNA. A still further aspect of the invention lies in a method ofpreparing a nitroreductase enzyme, comprising expressing a gene in abacterial cell, wherein the gene codes for a nitroreductase enzyme ofthe invention.

[0046] In an example of the invention described below in more detail,the gene expressed is a Bacillus gene or is a gene obtained bysubstitution, deletion and/or addition of nucleotides in or to aBacillus gene.

[0047] The invention also provides the use of a nitroreductase accordingto any of the aspects of the invention in manufacture of a medicamentfor anti-tumour therapy, and the use of a compound comprising anitroreductase according to any aspect of the invention conjugated to atargeting moiety in manufacture of a medicament for anti-tumour therapy.

[0048] The invention is now illustrated by the following specificexamples and in the accompanying sequence listing in which:

[0049] SEQ ID NO: 2 is a nitroreductase from B. amyloliquefaciens (codedfor by SEQ ID NO: 1) and designated “Bam YrwO”;

[0050] SEQ ID NO: 4 is a nitroreductase from B. subtilis (coded for bySEQ ID NO: 3) and designated “Bs YwrO”;

[0051] SEQ ID NO: 6 is a nitroreductase from B. subtilis (coded for bySEQ ID NO: 5) and designated “YrkL”;

[0052] SEQ ID NO: 8 is a nitroreductase from B. subtilis (coded for bySEQ ID NO: 7) and designated “YdeQ”;

[0053] SEQ ID NO: 10 is a nitroreductase from B. subtilis (coded for bySEQ ID NO: 9) and designated “YdgI”;

[0054] SEQ ID NO: 12 is a nitroreductase from B. subtilis (coded for bySEQ ID NO: 11) and designated “YodC”;

[0055] SEQ ID NO: 14 is a nitroreductase from E. coli (coded for by SEQID NO: 13) and designated “YabF”

[0056] SEQ ID NO: 16 is a nitroreductase from E. coli (coded for by SEQID NO: 15) and designated “YheR”;

[0057] SEQ ID NO: 17 is a nitroreductase from H. influenzae;

[0058] SEQ ID NO: 18 is a nitroreductase from T. aquaticus;

[0059] SEQ ID NO: 19 is a nitroreductase from Synechocystis sp PCC 6803;

[0060] SEQ ID NO: 20 is a nitroreductase from A. fulgidus;

[0061] SEQ ID NO: 21 is a nitroreductase from A. fulgidus.

[0062] SEQ ID NO: 23 is a nitroreductase from Campylobacter jejuni(coded for by SEQ ID NO: 22);

[0063] SEQ ID NO: 25 is a nitroreductase from Porphyromonas gingivalis(coded for by SEQ ID NO: 24);

[0064] SEQ ID NO: 27 is a nitroreductase from Yersinia pestis (coded forby SEQ ID NO: 26); and

[0065] SEQ ID NO: 29 is a nitroreductase from Helicobacter pylori (codedfor by SEQ ID NO: 28).

[0066] The invention is also illustrated by reference to theaccompanying Tables and FIGS. 1 to 4, in which:—

[0067]FIGS. 1 and 2 show sequence comparisons as set out in more detailin Example 8;

[0068]FIG. 3 shows enhancement of cytotoxicity of CB 1954 using enzymesof the invention; and

[0069]FIG. 4 shows enhanced toxicity of SN 23862 using enzymes of theinvention

EXAMPLE 1

[0070] A Nitroreductase Enzyme/Gene from Bacillus amyloliquefaciens

[0071] Briefly, extracts of Bacillus amyloliquefaciens were shown topossess nitroreductase activity. To purify this activity, crude cellextracts were subjected to ammonium sulphate, fractionation and anionexchange chromatography. The purified material was subject to N-terminalamino acid sequence analysis and the information obtained used to clonedthe gene via a PCR-based strategy. Following determination of itsnucleotide sequence the gene was overexpressed in E. coli and theresultant recombinant protein purified and characterised see table 1.

[0072] This analysis showed that the enzyme had properties which weredistinct from that of E. coli NfnB. Thus the protein had a morefavourable K_(m) for CB1954 (1.5-fold lower than the E. coli B NfnB) andfurthermore converted CB1954 into the 4HX form alone. It also differedfrom the E. coli B NfnB in that the enzyme showed no activity againstthe prodrug SN23862.

[0073] The isolated enzyme/gene represents a significant improvementover the E. coli NfnB enzyme with respect to its activity against theprodrug CB1954 ie., it produces only the 4HX derivative and has animproved K_(m) for CB1954.

[0074] A comparison of the amino acid sequence of the isolated enzymerevealed that it shared a very low level of homology to the rat DTD (c.25%), but exhibited high homology (70% sequence identity) with thepredicted product of a gene that has been discovered in the Bacillussubtilis genome sequencing project, designated ywrO. On this basis, wehave designated the cloned Bacillus amyloliquefaciens gene ywrO, and itsencoded enzyme YwrO.

[0075] YwrO BAM is a tetrameric flavoprotein (monomeric molecular massapproximately 22.5 kDa by SDS-PAGE, native molecular mass approximately90 kDa by gel filtration). Although it shares sequence homology with ratDTD it differs in its enzymic properties in that it can use only NADPHas cofactor (K_(m) 40 μM). In common with DTD it can reduce CB1954 butnot SN23862, reduction of CB1954 resulting in formation of the 4HXproduct only (K_(m) 617 μM, k_(cat) 8.2). It shows a high affinity forthe quinone menadione (K_(m) 3.4 μM) and has azoreductase and flavinreductase activity (K_(m) for FMN 53 μM, K_(m) for FAD 209 μM).

[0076] In more detail, N-terminal amino acid sequencing of the purifiedBacillus amyloliquefaciens nitroreductase enzyme resulted in thefollowing sequence,Met-Lys-Val-Leu-Val-Leu-Ala-Val-His-Pro-Asp-Met-Glu-Asn-Ser-Ala-Val-Asn.When this sequence was used to search available protein databases stronghomology was noted with the predicted amino acid sequence of ahypothetical protein, YrkL, identified in the Bacillus subtilis genomesequencing project. Significant homology was also evident with twoproteins, YabF and YheR, identified during the course of thedetermination of the Escherichia coli genome. These three hypotheticalproteins shared weak homology with a number of mammalian quinonereductases and NAD(P)H-oxidoreductases, such as the rat DTD.

[0077] In view of this observation, a strategy was formulated wherebysequence homology between the identified bacterial proteins, togetherwith the determined N-terminal amino acid sequence of the discoveredBacillus amyloliquefaciens enzyme, was used to amplify a region of thedesired encoding gene from the Bacillus amyloliquefaciens genome. Theone primer utilised in PCR was a degenerate oligonucleotide sequencewhich corresponded to a DNA sequence capable of coding for theN-terminal octa-peptide Val-His-Pro-Asp-Met-Glu-Asn. It was composed ofthe following nucleotides, 5′-GTNCAYCCNGATATGGARAA-3′, where Y indicatesthe presence of a T or C, R indicates the presence of A or G, and Nindicates the presence of either T, C, G or A. The second primer wasbased on the hypothetical sequence His-Gly-Trp-Ala-Tyr-Gly which wasfound to be entirely conserved between the hypothetical bacterialproteins YrkL (Bacillus subtilis) and YabF (E. coli), and partiallyconserved in YheR (E. coli). The degenerate oligonucleotide mixturesynthesised corresponded to the antisense DNA coding strand, viz.,5′-CCRTANGCCCANCCRTG-3′. E. coli YheR (90-95) Arg Gly Phe Ala Ser Gly E.coli YabF (84-89) His Gly Trp Ala Tyr Gly B. subtilis YrkL (85-90) HisGly Trp Ala Tyr Gly

[0078] The two primers were employed in PCR using chromosomal DNAisolated from Bacillus amyloliquefaciens and an amplified DNA fragmentof the expected size (approximately 230 bp) obtained. This was clonedinto plasmid pCR2.1 TOPO (Invitrogen) and its nucleotide sequencedetermined. Translation of the sequence obtained demonstrated thepresence of an open reading frame which encoded a polypeptide whichshared 66% sequence similarity with YrkL.

[0079] To obtain the entire structural gene, an approach was employedbased on inverse PCR. In essence, B. amyloliquefaciens DNA was cleavedwith the restriction enzyme StyI and the fragments generatedcircularised through their subsequent incubation with DNA ligase. Theligated DNA was then used as the template for a PCR employing twodivergent primers based on the sequenced 220 bp fragment. These wereBamNTR11 (5′-GCTTATTGACCGCTGAG-3′) and BamNTR14(5′-GTACAGTGCGCCTCCGC-3′). A 2.9 kb fragment was generated, cloned intopCR2.1 TOPO (Invitrogen) and the sequence of the insert determined. Thisallowed the identification of the nucleotide sequence of the remainingparts of the B. amyloliquefaciens gene. Using this information, acontiguous copy of the entire structural gene was amplified from the B.amyloliquefaciens chromosome using primers which encompassed thetranslational start codon (5′-GGTGTGATACATATGAAAGTATTG-3′) and resided3′ to the translational stop codon (5′-CGGGGATTCGAATTCTTTCTCAGG-3′). Theprimer at the 5′-end of the gene was designed such the sequenceimmediately 5′ to the ATG start codon became CAT. This change created anNdeI restriction site (CATATG), thereby allowing the cloning of the geneinto the equivalent site of the expression vector pMTL1015. Thismanipulation facilitated the subsequent overexpression of the gene, asinsertion of the gene at this point positions the start codon at anoptimum distance from the vector borne ribosome binding site.

[0080] The strategy employed to clone the BM YwrO gene could besimilarly employed to clone further genes encoding novelnitroreductases. This would involve purifying the desired enzymeactivity from a cell lysate, and then determining the N-terminalsequence. The data obtained could then be used to design anoligonucleotide primer corresponding to the sense strand of the DNAencoding part or all of the determined amino acid sequence. This primercould then be used, in conjunction with a second primer, to amplify partof the gene encoding the nitroreductase from the chromosome of thebacterial host using PCR. The second primer would correspond to theantisense strand of an internal portion of the targeted gene. Its designwould be based on regions of homology which are conserved amongst thetype of nitroreductase family that is sought. Thus, in the case of theDTD-like family, the oligonucleotide would, for example be based on theconserved motif His-Gly-Trp-Ala-Tyr-Gly (ie., amino acid residues 85-90in the BS YrkL protein). In the case of the NfnB-like family, theoligonucleotdie could be based on the motif Glu-Arg-Tyr-Val-Pro-Val-Met(ie., amino acid residues 170-176 in the BS YodC protein).

[0081] Such amplified fragments could then be cloned and sequenced, andnew primers designed based on this sequence to isolate the flankingregions of the gene by PCR. Once these have been cloned and sequenced,the entire, contiguous structural gene may be amplified using primerswhich extend beyond the 5′ and 3′ end of the translational start andstop codons.

[0082] Cloning of genes encoding novel nitroreductases may also beachieved without recourse to N-terminal sequencing of the enzyme, oreven its purification. This would involve basing the sequence of both ofthe oligonucleotides used in the initial PCR reaction on amino acidsequence motifs conserved amongst the two identified nitroreductasefamilies. Thus, in the case of the NfnB-like family, a sense primer(eg., 5′-ATTTCTAAAGAAGAGCTGACGGAA-3′) based on the motifIle-Ser-Lys-Glu-Glu-LeuI-Thr-Glu (ie., amino acid residues 13 to 20 ofBS YodC) could be employed with an antisense primer (eg.,5′-CATTACCGGTACATAGCGTTC-3′) based on the sequence motifGlu-Arg-Tyr-Val-Pro-Val-Met (ie., amino acid residues 170 to 176). Inthe case of the DTD-family a sense primer (eg.,5′-CATCCGGATATGGAAAAT-3′) based on the motif His-Pro-Asp-Met-Glu-Asn(ie., amino acid residues to 9 to 14 of BM YwrO) could be employed withthe an antisense primer (eg., 5′-TCCATATGCCCATCCATA-3′) based on thesequence motif Tyr-Gly-Trp-Ala-Tyr-Gly (ie., amino acid residues 85 to90). Once amplified, the rest of the gene could be isolated using thesame procedure as outlined above.

EXAMPLE 2

[0083]Bacillus subtilis Nitroreductases

[0084] As indicated above in Example 1, comparative analysis of the B.subtilis genome sequence with the amino acid sequence of the isolated B.amyloliquefaciens enzyme demonstrated the existence of an enzyme (YwrO)which shared 70% sequence identity. Unexpectedly, B. subtilis was foundto possess two homologues, YrkL and YdeQ, which share 54% and 51%sequence homology, respectively, with the B. amyloliquefaciens enzyme.All three enzymes share no homology with the E. coli B NfnB. They do,however, exhibit weak similarity (c. 25%) to the rat DT-Diaphorase(DTD). Whilst these proteins share a low level of sequence similarity toDTD, and other mammalian equivalents, they are characteristicallysmaller. This is because of the absence of an extensive internal proteindomain at the N-terminus of the protein. Thus, the functional equivalentdomain of the rat DTD between amino acid residues 51 to 82, are missingfrom the BM YwrO protein. In addition, the rat DTD has an extraCOOH-terminal domain. These bacterial enzymes are thus distinct fromtheir mammalian equivalents.

[0085] A further analysis of the B. subtilis genome, demonstrated thattwo homologues of the E. coli NfnB gene were present. Their encodedproteins (YdgI and YodC) share a barely detectable level of sequenceconservation with EC NfnB, of around 20% sequence identity.

[0086]Bacillus subtilis was thus found to carry at least 5 differentenzymes with nitroreductase activity. These are split into two families,thus;— DTD-like- 3 members:- YwrO, YrkL, YdeQ NfnB-like- 2 members:-YdgI, YodC

EXAMPLE 3

[0087] Recombinant Production of Nitroreductases from Bacillus subtilis

[0088] The DNA encoding all 5 B. subtilis nitroreductase enzymes werecloned from genomic DNA using PCR and the resultant genes, followingauthentification by nucleotide sequencing, subcloned into a proprietyCAMR expression vector (pMTL1015). The expression clones generated havebeen used to overproduce each of the 5 proteins and the enzymic activityof each assessed in crude lysates. This analysis has demonstrated thatwhilst the B. subtilis YwrO shares similar properties to the B.amyloliquefaciens homologue (ie., converts CB1954 to the 4HX derivativealone, but is inactive against SN23862), YrkL and YdeQ have no activityagainst either of the two prodrugs tested (CB1954 or SN23862) but theymay be active against other prodrugs.

[0089] Despite the extremely limited sequence similarity to EC NfnB,YdgI and YodC are active against both CB1954 and SN23862. They do,however, produce both the 2HX and 4HX derivatives of CB1954. Theircharacterisation has shown that they turn over CB1954 at higher ratesthan EC NfnB (YodC k_(cat) 58, YdgI k_(cat) 30.3 cf 6 for NfnB). Bothshow a high affinity for menadione and flavins, but they differ in thatwhereas YdgI uses both NADH and NADPH, YodC shows a preference for thelatter. The native molecular mass of YodC (approximately 90 kDa)indicates that it is tetrameric (molecular mass estimated from aminoacid sequence and by SDS-PAGE being approximately 22 kDa) whereas YdgIappears to be a dimer in the native state (molecular mass by gelfiltration approximately 49 kDa).

[0090] These finding are further illustrated in Table 2.

EXAMPLE 4

[0091]Bacillus lautus & Bacillus pumilis Nitroreductases

[0092] From 103 soil sample isolates tested, two strains (Bacilluspumilis CP044 and Bacillus lautus CP060) had been previously chosen aspossessing extracts which showed the most rapid reduction of both CB1954and SN23862. Purification experiments demonstrated that the activity inboth extracts was distributed across three distinct peaks. The presenceof more than one enzyme activity is consistent with our discovery ofmultiple forms of proteins in Bacillus able to turnover prodrugs.Eventual purification of the three enzymes of B. pumilis CPO44 revealedthat no one candidate exhibited properties which were an improvement onthe E. coli NfnB enzyme. In contrast, the proteins in peak 1 and peak 3of the B. lautus CP060 were determined to offer advantage over NfnB.

[0093] Thus, whilst the enzyme in peak 1 did not produce the required4HX derivative of CB1954, it exhibited a 4-fold lower K_(m) with theprodrug SN23862. The enzyme of peak 3 was, however, deemed to be ofgreatest value as it converted CB1954 solely into the 4HX derivative andhad a K_(m) approximately 4-fold lower than NfnB. Furthermore, it alsohad activity against SN23862. In this respect it shares the propertiesof both the Bacillus DTD-like family (ie., it produces only the 4HXderivative) and the NfnB-like family (ie., it is active againstSN23862)—these findings are illustrated in Table 3.

EXAMPLE 5

[0094] N-Terminal Sequencing of B. lautus Nitroreductase

[0095] Electrophoretic separation of the peak 3 demonstrated that 4protein bands were present which could account for the observed prodrugactivity. All four were subjected to N-terminal amino acid sequencingand the activity localised to the fourth protein band from which thenitroreductase may be purified.

EXAMPLE 6

[0096] Detection of Nitroreductase Activity in Thermophile Extracts

[0097] As an alternative source novel enzymes, a preliminary screen ofCAMRs thermophile collection was undertaken. Enzymes from this sourcemay have the advantage of greater stability, and therefore longevity ofaction. Strains were selected on the basis either of sensitivity toCB1954, or those which are resistant but which impart a yellow/goldencoloration to agar containing prodrug.

[0098] Two of these strains (B. thermoflavus and B. licheniformis)generated the cytotoxic 4HX form and were selected for further study.

EXAMPLE 7

[0099] Identification of Further Nitroreductase Enzymes

[0100] Having identified the two families of nitroreductase in Bacillus,a search was undertaken of both finished and unfinished genomes forhomologues, using YwrO and YodC/NfnB. On the basis of this searchhomologues of YwrO were identified in the genomes of Yersinia pestis andPorphyromonas gingivalis, and homologues of NfnB in the genomes ofPyrococcus furiosus, Haemophilus influenza, Synechocystis PCC 6803,Campylobacter jejuni, Archaeglobus, Helicobacter pylori, Heliocbacterfulgidus and Thermus aquaticus.

[0101] In addition to the above, two E. coli genes were found to behomologues of rat DTD and YwrO, and were designated Yher and YabF. Theywere discovered to share the characteristic of YwrO in that they lackthe internal protein domain found in the rat DTD enzyme and functionalmammalian homologues.

[0102] (i) P. gingivalis YwrO Homologue

[0103]P. gingivalis YwrO homologue is a dimeric flavoprotein with nativemolecular mass estimated by gel filtration at 40 kDa. Although it sharessequence homology with DTD and forms only the 4HX reduction product ofCB1954 (K_(m) 1200 μM, k_(cat) 3.2), it differs from DTD in that it isactive with SN23862 and it can only use NADH as cofactor (cf DTD whichcan use either NADH or NADPH and is inactive with SN23862). It canreduce azodyes but it is inactive with menadione or flavins.

[0104] (ii) C. jejuni NfnB Homologue

[0105]C. jejuni NfnB homologue produces only the 4HX reduction productof CB1954 (K_(m) 143 μM, k_(cat) 11.2) using NADPH as cofactor and it isalso active with SN23862. It can use the quinone menadione as substrateas well as azodyes and the flavins FMN and FAD.

[0106] (iii) Archaeoglobus fulgidus NfnB Homologue

[0107]Archaeoglobus fulgidus NfnB homologue is a dimeric flavoprotein of42 kDa native molecular mass, producing the 4HX derivative of CB1954only (K_(m) 690 μM, k_(cat) 56.2) using NADPH as cofactor. It is alsoactive with SN23862 and menadione (K_(m) 9 μM), but does not decolouriseazodyes and has only weak flavin reductase activity.

[0108] (iv) H. influenzae and H. pylori NfnB Homologues

[0109] Both these enzymes are dimeric flavoproteins and form the 4HXreduction product of CB1954 using NADPH in preference to NADH, but haveno activity with azodyes. The former also lacks activity with thequinone menadione and flavins FMN or FAD. Both however have weakactivity with SN23862 and may be active with other prodrugs.

[0110] (v) Y. pestis nfnB Homologue and Synechocystis YwrO Homologue

[0111] Both these proteins reduce CB1954 but produce only the relativelynon-toxic 2HX derivative using NADPH as cofactor. They do however showactivity with SN23862 and the former can also reduce azodyes.

EXAMPLE 8

[0112] Comparison of Nitroreductase Sequences

[0113] We compared the amino acid sequences of nitoreductases accordingto the invention with each other and with known rat, human and E. colisequences, and the results are illustrated in FIGS. 1 and 2. In FIG. 1,rat, mouse and two human sequences make up the first four lanes forcomparison purposes. It is evident that nitroreductases of the inventionare lacking a sequence from positions 51-82 of the rat sequence.

[0114] In FIG. 2, sequences of nitroreductases of the invention arecompared with the known E. coli sequence, which is designated nfmB inthe second-to-last lane.

EXAMPLE 9

[0115] Materials and Methods

[0116] Reagents

[0117] CB 1954 and SN 23862 were generous gifts from Dr D. Wilman,Institute of Cancer Research, Sutton, Surrey and Prof. W. Denny,University of Auckland, New Zealand, respectively. DMSO was obtainedfrom Aldrich. Restriction endonucleases were from NBL and New EnglandBiolabs. T4 DNA ligase was from Boehringer Mannheim, Taq polymerase fromBioline and native Pfu polymerase from Stratagene. DNA purificationreagents were from Cambio (Gene Releaser™ resin), Promega (Wizard™plasmid purification resin) and Qiagen (PCR clean-up kit andgel-extraction kit). PCR-BluntII-TOPO was obtained from Invitrogen.Deoxyribonucleotides were from Fermentas. Oligonucleotides weresynthesized by CAMR Structural Sciences. DNA labelling reagents werefrom Amersham (ECL kit). All other reagents from Sigma Chemical Co.Ltd

[0118] Enzymes

[0119]E. coli B nitroreductase was purified as previously described (1).All other recombinant enzymes were purified by anion exchangechromatography except for the NfnB homologue of C. jejuni (for detailssee Results section).

[0120] Enzyme Assays

[0121] Quantitative assays using CB 1954 or SN 23862 as substrate werecarried out at 37° C. by HPLC as previously described (2) (3). Whenqualitative assays were used to identify column fractions the standardconditions were as follows: 1 mM prodrug, 2 mM NAD(P)H, 4% DMSO in 100mM sodium phosphate buffer pH 7, 37° C. Incubation times variedaccording to the enzyme activity being studied. Assays using menadioneas substrate were carried out spectrophotometrically as previouslydescribed (1) using cytochrome c as terminal electron acceptor andsimilar procedures were used to assay flavin reductase activity with FMNand FAD as substrate and with cofactors NADH and/or NADPH. Azoreductaseactivity was assessed qualitatively by incubating aliquots of enzymeswith 500 μl o-methyl red or p-methyl red (200 μM in 20 mM BisTris pH7.0, 1% DMSO)+NADPH (500 μM) as cofactor at 37° C. and noting the timeto decolourisation.

[0122] Protein Assay

[0123] Protein content of samples was estimated by BCA assay (Pierce)

[0124] Electrophoresis

[0125] Homogeneity of purified proteins was assessed in SDS or nativePAGE using a Phastsysytem (Amersham Pharmacia) according to themanufacturer's instructions or in BioRad precast Ready Gels.Visualisation of the separated proteins was achieved using CoomassieBlue (Phastsystem) or silver staining (BioRad) as appropriate.

[0126] Micro-Organisms

[0127] Organisms were sourced as shown in Table 5.

[0128] Amplification & Cloning of DNA

[0129] PCR templates were prepared from bacterial colonies or cellpastes using Gene Releaser resin. Typically, a single colony wasvortexed for 20 seconds in 28 μl Gene Releaser slurry+28 μl diluent in a500 μl polypropylene tube and heated for 6 min in a 750W microwave ovenat full power. The resin was then pelleted and the supernatant dividedinto 5 μl aliquots for direct use in PCR reactions. Where DNA of greaterpurity was required, chromosomal DNA was prepared by standard methods.PCR products intended for cloning and expression were generated using 8UPfu polymerase, 5 μl native Pfu buffer, 120 nmol⁻¹ forward and reverseprimers and 200 μmol⁻¹ of each deoxyribonucleotide in 50 μl total volumein 200 μl thin-wall tubes in a Perkin Elmer PCR9600 thermocycler.Otherwise Taq polymerase was used in place of Pfu. Where necessary,forward primers contained mismatches to introduce restriction enzymesites through the start codons of amplified genes. Pfu PCR products wereligated into pCR-BluntII-TOPO according to the supplier's instructionsand transformed into E. coli. Subsequent DNA manipulation followedstandard methods.

[0130] Overexpression in E. coli

[0131] Genes subcloned into pMTL1015 (Alldread, R. M.; Nicholls, D. J.;Murphy, J. P.; Atkinson, M. A.; Scawen, M. D.; Atkinson, T. & Sundaram,T, K., Escherichia coli malate dehydrogenase gene expression system:characteristics and use as a hyper-expression cassette, unpublished)were expressed in E. coli HMS174.

[0132] Overnight cultures of 50 ml were made up to 1 litre with L-brothcontaining 10 μmol ml⁻¹ tetracycline and grown at 37° C. for a further24 hours with slow shaking (˜150 rpm) in non-baffled flasks. Genessubcloned into pET21b(+) or pET21d(+) were expressed according tostandard methods (4) using E. coli NovaBlue (DE3) as the host. For pETexpression aeration was maximized by the use of baffled flasks andanti-foaming agents.

[0133] Amplification of nfnB and ywrO Homologues by Reverse PCR

[0134] 200 ng of StyI-cut or HindIII-cut genomic DNA was self-ligatedovernight at 14° C. with 2·5 units T4 DNA ligase in a volume of 50 μl inthe buffer supplied with the ligase. Circularized fragments containingthe target gene were then amplified by PCR using a pair ofoutward-facing primers based on a previously cloned 211 bp fragment.After agarose-gel electrophoresis of completed PCRs, ligase-dependentbands of the expected size were excised from the gel and purified

[0135] Sequence Analysis

[0136] DNA sequencing was performed by CAMR Structural Sciences or Oswelusing plasmids eluted in water from Wizard resin, or PCR products elutedin water from PCR clean-up columns. Electropherogram-editing and contigassembly were done using Sequence Manager (LaserGene). Early-releasegenome databases were searched using the Sanger Centre's gapped-TBLASTAserver and the gapped-TBLASTX server at NIH. GenBank was searched usingthe TFastA program within GCG version 10. Multiple alignments weregenerated with the PileUp program also within GCG PROGRESS

[0137] In vitro Cytotoxicity Tests

[0138] Microtitre plates (96 well) were obtained pre-seeded with V79cells at 10,000 cells/ml (European Collection of Animal Cell Cultures,ECACC) in DMEM+10% foetal calf serum. All treatments were prepared onduplicate plates and transferred to the cells prior to adding enzyme (10μl) to the appropriate wells. CB 1954 was dissolved in DMSO (Sigma,tissue culture grade) so that the appropriate concentrations could bedispensed by adding 5 μl per well. NAD(P)H was dissolved in sterile PBSto give the appropriate final concentration by adding 10 μl per well.Enzymes were diluted in sterile PBS. All aqueous solutions were filtersterilised before use and operations carried out aseptically in alaminar flow hood. The cells were exposed for 3 h to CB 1954 or SN 23862(3.9-500 μM in doubling dilutions) alone or in combination with cofactor(NAD(P)H 125 or 250 μM) and enzyme (4 μg) by removing the growth mediumand replacing it, after washing twice with PBS, with 200 μl serum freemedium containing the various reaction components. After exposure thecells were washed with PBS, fresh medium with serum was added and theplates were left to incubate at 37° C. and 5% CO₂ for 3-4 days untilcells in control untreated wells had achieved confluent growth.Cytotoxicity was quantified by sulphorhodamine B (SRB) assay. Briefly,the growth medium was removed and the cells fixed by addition of 100 μlper well of cold 10% TCA for 30 min. The TCA was removed and the fixedcells washed with water before adding 100 μl per well of 0.4% dye in 1%acetic acid and incubating at room temperature for 30 min. Excess dyewas removed and the wells washed 4 times with 1% acetic acid. After airdrying at room temperature the dye was solubilised by adding 100 μl of10 mM Tris to each well and shaking for 15 min. The plates were read at492 nm in a Titertek plate reader. Cytotoxicity towards treated cellswas expressed as % of A₄₉₂ of untreated controls and statisticalanalysis was performed using the Mann-Whitney test. ED₅₀'s werecaculated using probit analysis.

[0139] Antisera to Recombinant Enzymes

[0140] Polyclonal antisera were raised against recombinant enzymes byimmunising rabbits with 0.1 μg of protein in 50% PBS:50% Freund'scomplete adjuvant, total volume 400 μl. Blood was collected by ear bleedafter 6-8 weeks and again 10 days after a booster immunisation ofprotein, and titre assessed by ELISA. 96-well plates were coated with 1μg ml⁻¹ of the proteins and blocked with 10% v/v foetal calf serum(FCS), antibodies were diluted in assay buffer (1% FCS) and applied indoubling dilutions across the plate from 1 in 100 to 1 in 51 200 or 1 in150 to 1 in 76 800. A secondary antibody conjugated with HRP (dilution 1in 10 000) was used to develop the assay with3,3′,5,5′-tetramethylbenzidine (TMB) as substrate.

[0141] Western Blots

[0142] Recombinant enzymes were run on SDS PAGE 4-20% gradient at 0.05,0.5, 5, 50 and 500 ng with 500 ng of the other antigens to testcross-reactivity. The antisera were added at a dilution of 1/10 000 or1/20 000. Secondary antibody dilutions were as shown above and detectionwas by ECL (Amersham).

[0143] Results and Discussion

[0144] Overexpression in E. coli of all Nitroreductase Genes

[0145] Of the 15 genes identified, 14 were overexpressed and this workwas reported in the annual report for Project 650 in March 2000.Subsequently it was discovered that the enzymes of the thermophileArchaeoglobus fulgidus were in fact relatively inactive, and plans toclone the Thermus thermophilus gene were abandoned.

[0146] A total of six recombinant enzymes were selected for furtherstudy and, together with the E. coli NfnB enzyme, purified to allowantibody preparation as reported previously (Annual report, project 650,2000). It has not proved possible to purify the Helicobacter pylorienzyme. However, as this enzyme produces a mixture of the 2 and 4HX withCB 1954, and exhibits relatively low activity, attempts to purify itwere abandoned. The Campylobacter jejuni enzyme and the Bacillussubtilis enzymes YrkL, YdeQ and YwrO have now been purified.

[0147]C. jejuni NfnB

[0148] This enzyme overexpressed in E. coli was purified by anionexchange chromatography using 15 mM piperazine pH 10.0 followed by gelfiltration.

[0149] YwrO

[0150]E. coli bearing pET21 b(+) with ywrO inserted between the EcoR1and Nde1 sites was grown in 2YT (300 ml+50 μg ml⁻¹ ampicillin).Expression of the YwrO protein was induced by addition of IPTG (200 μgml⁻¹). The protein was then purified from crude extracts by anionexchange chromatography (FPLC, Mono Q) in 20 mM Tris pH 7.5. Initially,since the substrate specificity of the protein was unknown and it wasinactive with either CB 1954 or SN 23862, fractions were identified onthe basis of the mobility of the overexpressed protein using SDS-PAGE.Subsequently, active fractions were identified by decolourisation of theazodye, o-methyl red.

[0151] YdeQ

[0152] YdeQ was purified from a 1.5 ml post-induction (IPTG) lysate byanion exchange chromatography (FPLC Mono Q) in 20 mM Tris pH 7.5.Detection of the presence of the correct protein in fractions elutingfrom the column on a salt gradient (0-500 mM KCl) was by mobility onSDS-PAGE as for YwrO, since there was no activity with prodrugs.

[0153] YrkL

[0154] YrkL was purified from crude extracts of an IPTG-induced cultureby anion exchange chromatography in 20 mM Tris pH 7.6 and fractionsidentified by decolourisation of azodye as described for YwrO above

[0155] Determination of Polyclonal Antisera Titres by ELISA

[0156] Antisera to E. coli B NfnB and six novel proteins (Haemophilusinfluenzae and Campylobacter jejuni NfnB homologues, YdgI and YodC ofBacillus subtilis, YwrO of B. amyloliquefaciens and Porphyromonasgingivalis YwrO homologue) have been raised in rabbits by inoculationwith 100 μg of protein in PBS+ Freund's complete adjuvant. Titres wereassessed by ELISA after 8 weeks, and 10 days after a booster inoculationof a further 100 μg of protein per rabbit. Pre-immune sera were alsotested. TABLE 5 Novel nitroreductases, rabbit polyclonal antisera titresDilution Secondary Pre-boost Post-boost Antiserum range Ab dilutiontitre (50%) titre E. coli BNfnB 150-76800 10000 1300 11000 P. gingivalisYwrO  50-25600 5000 1000 3100 YwrO BAM  50-25600 5000 1200 2800 YdgI BS150-76800 2000 4900 25000 H. influenzae NfnB  20-10240 10000 180 760YodC BS 100-51200 10000 3700 7500 C. jejuni NfnB  50-25600 5000 11002500

[0157] 96 well plates were coated overnight at 4° C. with the antigensat 1 μg/ml. After washing and blocking antisera were added in doublingdilutions in an appropriate range across the plates. The plates weredeveloped using goat anti-rabbit IgG peroxidase conjugate with TMB assubstrate, and read at 450 nm.

[0158] Pre-immune sera showed little or no binding to the antigens.Antisera were aliquoted and stored at −20° C.

[0159] Western Blots

[0160] Antigens were run on SDS PAGE 4-20% gradient at 0.05, 0.5, 5, 50and 500 ng with 500 ng of other antigens to test cross-reactivity. Theantisera were added at a dilution of 1/10000 (α-P. gingivalis YwrO,α-YwrO BAM and α-H. influenzae NfnB) or 1/20000 (α-E. coli B NfnB andα-YdgI). Secondary antibody dilutions were as shown above and detectionwas by ECL.

[0161] Results are shown in FIGS. 3(a-e) E. coli B NfnB could bedetected at 5 ng in this system (FIG. 3b) and no cross-reactivity wasdetected with the other antigens, whereas P. gingivalis YwrO, YwrO BAMand YdgI were detected only at 500 ng, but also with nocross-reactivity. However, antiserum raised against YdgI (FIG. 3d)showed a degree of cross-reactivity with 500 ng of both E. coli B NfnBand YodC, whilst detecting YdgI at 50 ng. Sequence similarity betweenNfnB and the B. subtilis enzymes is low and the results suggest agreater degree of structural similarity may exist between them.

[0162] Kinetics of Each Recombinant Product Against CB1954 and OtherProdrugs TABLE 6 Novel nitroreductases: physical characterisation andsubstrate specificities Prodrug activation CB 1954 K_(m) k_(cat) MW SN23862 Quinone Azo- Flavin Enzyme product CB 1954 (s⁻¹) (kDa) acitvityCofactor reductase Reductase reductase C. jejuni 4HX 217 6.1 Monomer223.7/6.4 NADPH Yes Yes Yes 24 P. gingivalis 4HX 1200 3.2 Dimer Yes NADHNo Yes No ˜42 (weak) H. influenzae 4HX 690 56.2 ˜36 3365/39.8 NADPH YesNo Weak YodC 4HX 552.2 58.0 Tetramer 957.3/53.1 NADPH Yes Yes Yes ˜95.5YdgI 4HX 3863.9 30.3 Dimer Yes NADPH Yes Yes Yes ˜49 H. pylori 4 > 2HXND ND Monomer Weak NADPH ND No ND 24 Y. pestis 2HX ND ND Monomer WeakNADPH ND Yes ND 21.3 Synechocystis 2HX ND ND Monomer Weak NADPH ND No ND22.7 A. fulgidus 4HX ND ND ˜42 Yes NADPH ND ND ND 2267

[0163] Kinetics of the interaction between 5 novel enzymes and theprodrugs CB 1954 and SN 23862 have been estimated (see Table 6). Thestudy was restricted to those enzymes which produce solely the 4HXreduction product of CB 1954 (the nitroreductase of the thermophile, A.fulgidus although purified to homogeneity, proved to have only minimalactivity at 37C.)

[0164] SN 23862 Activity

[0165] Kinetic parameters for SN 23862 were assessed by HPLC assay anddetermined for YodC BS and 3 NfnB homologues. YdgI BS did not showMichaelis-Menten kinetics, the relationship between [S] and rate ofreaction being sigmoidal, suggesting an allosteric interaction.Modelling of the active site region may indicate how this proteindiffers from the highly related YodC. The crystal structure of NfnB isnow available and studies have commenced to model the active sites ofYodC, YdgI and H. influenzae NfnB homologue and their interaction withCB 1954 and NADPH. The rate of reduction of SN 23862 shown by P.gingivalis YwrO homologue was too slow for kinetic parameters to becalculated accurately.

[0166] Although the SN 23862 K_(m) for YodC is high, the k_(cat) is alsohigh, thus accounting for the cytotoxic action of the combination ofenzyme, cofactor and prodrug observed in V79 cells (FIG. 4).Additionally, although kinetic parameters could not be determined forYdgI, it is clear that the cytotoxic derivative of SN 23862 is producedat sufficiently high concentrations for cell killing to occur under theconditions used. TABLE 7 Substrate and cofactor specificity for YdgISubstrate Cofactor K_(m) (μM) k_(cat) (s⁻¹) k_(cat)/K_(m) Menadione NADH127.0 ± 10  628.0 ± 16.8 4.94 FMN NADH 158.0 ± 16 3002.0 ± 94.8 19.0NADPH  12.0 ± 1.4  345.2 ± 7.6 28.7 FAD NADH 150.0 ± 19.0 2580.7 ± 7917.2 NADH FMN 1 mM  59.0 ± 7.0 2258.1 ± 64.0 38.3 Menadione  6.6 ± 8.2 766.0 ± 24.0 116.1 NADPH Menadione 295.0 ± 29  96.0 ± 2.8 0.3 100 μM

[0167] Substrate Specificity

[0168] Activity of the B. subtilis enzymes YodC and YdgI with thequinone, menadione and with the flavins FMN and FAD with cofactors NADHand NADPH has been completed and the results are shown in Tables 7 and8. Assays were carried out spectrophotometrically at 37° C. in 10 mMTris pH 7.5 using cytochrome c as terminal electron acceptor.

[0169] Both these enzymes therefore are flavin reductases and quinonereductases, but in all cases the affinity of YodC for the substrates ishigher than that of YdgI. Although they are highly related in amino acidsequence, they differ in their cofactor specificity, YdgI showing adistinct preference for NADH, whereas YodC appears to be more like aDTD, showing similar rates of reaction with either cofactor. Both arepotently inhibited by dicumarol (as are DTD and NfnB), but the mechanismof inhibition differs. These results confirm the differences inproperties between the two proteins despite their sequence similarity.Substrate and cofactor specificity for YodC Substrate Cofactor K_(m)(μM) k_(cat) (s⁻¹) k_(cat)/K_(m) Menadione NADH 1.0 ± 0.1 415.4 ± 14.8415.4 NADPH 1.6 ± 0.2 329.5 ± 18.4 205.9 FMN NADH 0.5 ± 0.1 293.8 ± 19.7587.6 NADPH 1.0 ± 0.1 328.2 ± 6.4  328.2 FAD NADH 0.6 ± 0.1 269.0 ± 4.9 448.3 NADPH 2.4 ± 0.3 282.9 ± 7.4  117.9 NADH FMN 5 μM 205.0 ± 26.0 318.3 ± 11.0 1.6 Menadione 5 μM 178.0 ± 18.0  305.6 ± 21.2 1.7

[0170] The novel enzymes from C. jejuni and H. influenzae were alsocharacterised with respect to the flavins, menadione and cofactors andthe results shown in Tables 9 and 10. TABLE 9 Cofactor and substratespecificity for C. jejuni NfnB Substrate K_(m) k_(cat) k_(cat)/K_(m)Menadione  1.3 ± 0.2 μM 66.1 50.8 (1 mM NADPH) NADPH 69.6 ± 8.8 μM 62.50.9 (20 μM menadione) FMN  0.7 ± 0.2 μM 42.3 60.4 FAD  3.3 ± 0.4 μM 57.817.5

[0171] Both these enzymes show quinone reductase activity with highaffinity and distinct preference for NADPH as cofactor. The C. jejuniprotein is also a flavin reductase showing high affinity for both FADand FMN, but H. influenzae NfnB homologue shows little activity withthese substrates with either NADH or NADPH as cofactor. Like the B.subtilis enzymes, the former can reduce azodyes, but the latter shows noactivity with either o- or p-methyl red in quantitative assays. TABLE 10Substrate and cofactor specificity for H. influenzae NfnB SubstrateK_(m) k_(cat) k_(cat)/K_(m) Menadione 9.0 ± 0.6 μM 177.8 19.8 (1 mMNADPH) (1 mM NADH) 0.8 ± 0.2 μM 24.1 31.0 NADPH 2.9 ± 0.5 μM 154.2 53.2(menadione 100 μM)

[0172] Like its homologues in B. subtilis and B. amyloliquefaciens, theYwrO of P. gingivalis is an azoreductase, but it showed little activitywith menadione or flavins. Initial studies suggested that it may be anNADH oxidase, but further work is needed to determine its substratespecificity and possible physiological role. It is almost completelyinactive with NADPH.

[0173] Cytotoxicity Studies Against Cell Lines with Purified Enzymes andSelected Prodrugs

[0174] In vitro Cytotoxicity with CB 1954

[0175] Enhanced in vitro cytotoxicity against V79 cells of CB 1954 wasdemonstrated for NfnB, the YwrO of B. amlyoliquefaciens and the 5 othernovel proteins. Cytotoxicity was assessed by staining withsulforhodamine B 3-4 days post-treatment with prodrug, enzyme andcofactor. The H. influenzae NfnB homologue was the most potent, whilstthe YwrO homologues were the least potent of the novel enzymes. (FIG. 3)

[0176] In vitro Cytotoxicity (SN 23862)

[0177] In vitro cytotoxicity assays were carried out using thisalternative prodrug with NfnB and five of the novel enzymes (YwrO of B.amyloliquefaciens is inactive with this substrate) (FIG. 4).Dose-related cytotoxicity was seen with all the enzymes except for P.gingivalis YwrO homologue. The order of potency was similar to that withCB 1954 the most potent being the NfnB homologue of H. influenzae.Kinetic studies with P. gingivalis YwrO homologue and SN 23862 indicatethat the rate of reduction is very slow (see above) and this may explainthe lack of activity in cytotoxicity assays where a critical level ofthe cytotoxin is probably necessary for effective cell killing. TABLE 11ED₅₀'s for novel nitroreductases in in vitro cytotoxicity tests using CB1954 or SN 23862, calculated by probit analysis Enzyme ED₅₀ CB 1954 (μM)ED₅₀ SN 23862 YwrO BAM 137.1 — YdgI 15.3 76.6 YodC 20.3 74.2 NfnB 6.330.7 H. influenzae NfnB 4.7 17.1 C. jejuni NfnB 55.8 102.3 P. gingivalisYwrO 252.3 —

[0178] Pae3

[0179] The protein encoded by Pae3 was overexpressed in E. coli andpurified by anion exchange chromatography. It is most highly related tothe human form of DTD (cf other nitroreductase sequences used to searchdatabases) and in this context, it is perhaps not surprising that it isinactive with both prodrugs, CB 1954 and SN 23862. More unexpectedly, itis also inactive with flavins and azodyes, properties which are sharedby several members of the DTD/YwrO family of enzymes. However, it is aquinone reductase, and kinetic parameters were determined for thissubstrate using NADPH as cofactor, the rate of reaction using NADH ascofactor being approximately 5-fold lower. TABLE 12 Kinetic parametersfor Pae3 of Pseudomonas aeruginosa K_(m) k_(cat) K_(si) Menadione  3.8μM 153.4 s⁻¹ 18.0 μM NADPH 308.4 μM  33.8 s⁻¹ —

[0180] Efa1

[0181] This gene is more highly related in sequence to ydgI compared tothe other sequences used to search databases. The gene product of Efa1expressed in E. coli was purified by anion exchange chromatography in 20mM Tris pH 7.6 and its substrate specificity determined using theprodrugs, menadione and flavins (Table 13). The reduction of CB 1954resulted in the formation of both the 2 and 4HX products, in similarproportions to those formed by NfnB (approximately 50% of each productformed). SN 23862 reduction formed the 2HX cytotoxic product, butkinetic parameters were not determined for this substrate. It is aflavin, azo- and quinone reductase and shows a distinct preference forNADH as cofactor. Despite the sequence similarity to YdgI, therefore,the properties of the protein differ significantly (cofactorspecificity, product formation) indicating substantial differences instructure. TABLE 13 Kinetic parameters for Efa 1 of Enterococcusfaecalis Substrate K_(m) k_(cat) K_(si) CB 1954  4100 μM  12.0 s⁻¹Active ND ND — Menadione 107.4 μM 264.8 s⁻¹ 18.0 μM NADH (1 mM  44.3 μM314.9 s⁻¹ — FMN) FMN 104.3 μM 340.0 s⁻¹ — FAD 133.8 μM 187.8 s⁻¹ —

[0182] Smu2

[0183] The Smu2 gene shows sequence similarity to the nfnB homologue ofH. influenzae. The protein, overexpressed in E. coli Top 10 was purifiedfrom a crude extract by anion exchange chromatography in 15 mMpiperazine pH 10.0 (pI estimated from sequence to be 8.36). Like the“parent” protein it rapidly reduces CB 1954 with formation of the 4HXproduct only and it uses NADPH preferentially as cofactor. The cytotoxic2HX product was formed on reduction of SN 23862. With menadione assubstrate, it was virtually inactive with NADH. It is a potent quinonereductase but shows no activity with flavins, again resembling H.influenzae NfnB. TABLE 14 Kinetic parameters for Smu2 of Streptococcusmutans K_(m) k_(cat) CB 1954  2700 μM  96.4 s⁻¹ Menadione  2.7 μM 201.0s⁻¹ NADPH  1.3 μM 188.7 s⁻¹ (100 uM menadione)

[0184] Pmu2

[0185] This gene also shows sequence similarity to the nfnB of H.influenzae and, like Smu2 shares similar substrate and cofactorspecificity. It is a quinone reductase with high affinity and uses NADPHas cofactor, however it can use NADH but with a 2-fold decrease in rateof reaction (substrate menadione). It has little activity with flavinswith either cofactor. It forms the 4HX reduction product of CB 1954exclusively and has a greater affinity for this substrate than Smu2.With SN 23862 it forms the cytotoxic 2HX product, but kinetic parameterswere not determined. TABLE 15 Kinetic parameters for Pmu2 of Pasturellamultocida K_(m) k_(cat) CB 1954 692.4 μM  8.6 s⁻¹ Menadione  2.6 μM 23.5s⁻¹ NADPH  2.9 μM 24.6 s⁻¹ (25 uM menadione)

[0186] References

[0187] 1. Anlezark, G. M., R. G. Melton, R. F. Sherwood, B. Coles, F.Friedlos, and R. J. Knox. 1992. The bioactivation of5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB1954)—I. Purification andproperties of a nitroreductase enzyme from Escherichia coli—a potentialenzyme for antibody-directed enzyme prodrug therapy (ADEPT). BiochemPharmacol. 44(12):2289-95.

[0188] 2. Anlezark, G. M., R. G. Melton, R. F. Sherwood, W. R. Wilson,W. A. Denny, B. D. Palmer, R. J. Knox, F. Friedlos, and A. Williams.1995. Bioactivation of dinitrobenzamide mustards by an E. coli Bnitroreductase. Biochem Pharmacol. 50(5):609-18.

[0189] 3. Knox, R. J., M. P. Boland, F. Friedlos, B. Coles, C. Southan,and J. J. Roberts. 1988. The nitroreductase enzyme in Walker cells thatactivates 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB 1954) to5-(aziridin-1-yl)-4-hydroxylamino-2-nitrobenzamide is a form of NAD(P)Hdehydrogenase (quinone) (EC 1.6.99.2). Biochem Pharmacol. 37(24):4671-7.

[0190] 4. Studier F W, R. A. H., Dunn J J, Dubendorff J W. 1990. Use ofT7 polymerase to direct expression of cloned genes. Meth Enzymol.185:60-85.

[0191] 5. Zenno, S., T. Kobori, M. Tanokura, and K. Saigo. 1998.Conversion of NfsA, the major Escherichia coli nitroreductase, to aflavin reductase with an activity similar to that of Frp, a flavinreductase in Vibrio harveyi, by a single amino acid substitution. JBacteriol. 180(2):422-5.

[0192] 6. Zenno, S., H. Koike, M. Tanokura, and K. Saigo. 1996.Conversion of NfsB, a minor Escherichia coli nitroreductase, to a flavinreductase similar in biochemical properties to FRase I, the major flavinreductase in Vibrio fischeri, by a single amino acid substitution. JBacteriol. 178(15):4731-3.

[0193] The invention thus provides nitroreductase enzymes, DNA and genestherefor and methods of obtaining such enzymes and of using the enzymesand DNA coding therefor in clinical applications. TABLE 1Characteristics of nitroreductase enzymes from Bacillusamyloliquefaciens M. Wt CB1954 SN23862 ENZYME (Kda) Product Km kcat Kmkcat E. coli NCnB 24 2/4HX 862 6.0 2500 26.4 Rat DTD 33 4HX 826 0.07inactive inactive Bam YwrO 22 4HX 617 2.0 inactive inactive

[0194] TABLE 2 Characteristics of nitroreductase enzymes found in theBacillus subtilis genome DTD-like Family NfnB-like Family YwrO YrkL YdeQYdgI YodC Homology^(a) 70% 54% 51% 25% 24% CB1954 4HX inactive inactive2/4HX 2/4HX SN23862 inactive inactive inactive active active

[0195] TABLE 3 Fractionation of nitroreductase activity in cell extractsof Bacillus lautus and Bacillus pumilis ENZYME M. Wt CB1954 SN23862ACTIVITY (kDa) Product Km Km B. pumilis CP044 Peak 1 ND 4HX v. low NDPeak 2 ND 4HX >1000 ND Peak 3 ND 2/4HX    999 ND B. lautus CP060 Peak 135 2HX    211 325 Peak 2 42 4HX >2000 none Peak 3 47 4HX    257 active

[0196] TABLE 4 Characteristics of nitroreductase activity ofthermophiles identified as being sensitive to CB 1954 CB1954 SN23862STRAIN Product NADH NADPH NADH NADPH 1078 2/4HX 13.8 22.6 8.5 17.62122^(a) 2/4HX 36.6 56.0 33.4 62.8 6012^(b) 4 > 2HX 15.2 37.8 8.2 35.26013^(c) 2HX 9.8 49.4 6.4 39.0 6031^(d) 2HX 11.9 42.1 8.2 33.8 6036 2HX10.7 26.7 7.3 26.2 6044 2HX 4.0 21.3 4.5 9.9

[0197]

1 29 1 525 DNA Bacillus amyloliquefaciens CDS (1)..(525) 1 gtg aaa gtattg gta tta gcg gtt cac cct gac atg gag aac tca gcg 48 Met Lys Val LeuVal Leu Ala Val His Pro Asp Met Glu Asn Ser Ala 1 5 10 15 gtc aat aaggca tgg gca gaa gaa tta aaa aaa cat gat gaa ctc acg 96 Val Asn Lys AlaTrp Ala Glu Glu Leu Lys Lys His Asp Glu Leu Thr 20 25 30 gtc cgt gag ctttat aaa gaa tat ccg gac ggg caa atc gat gcg gaa 144 Val Arg Glu Leu TyrLys Glu Tyr Pro Asp Gly Gln Ile Asp Ala Glu 35 40 45 aag gaa cgt cag ctgtgt gaa cag tat gac cgg atc gta ttt caa ttt 192 Lys Glu Arg Gln Leu CysGlu Gln Tyr Asp Arg Ile Val Phe Gln Phe 50 55 60 ccg ctg tat tgg tac agtgcg cct ccg ctt tta aaa aca tgg atg gat 240 Pro Leu Tyr Trp Tyr Ser AlaPro Pro Leu Leu Lys Thr Trp Met Asp 65 70 75 80 cat gtg ctg tcg tac ggctgg gcc tac ggc tcc aaa gga aag gcg ctg 288 His Val Leu Ser Tyr Gly TrpAla Tyr Gly Ser Lys Gly Lys Ala Leu 85 90 95 cat ggc aaa gaa ttg atg ctggct gtt tcc gta ggt gcc gga gag gat 336 His Gly Lys Glu Leu Met Leu AlaVal Ser Val Gly Ala Gly Glu Asp 100 105 110 gca tac cag gca gga ggg tcaaac cac ttt aca ttg agc gag ctg tta 384 Ala Tyr Gln Ala Gly Gly Ser AsnHis Phe Thr Leu Ser Glu Leu Leu 115 120 125 agg ccg ttt cag gca atg gctaat ttt aca ggt atg acc tat ttg ccg 432 Arg Pro Phe Gln Ala Met Ala AsnPhe Thr Gly Met Thr Tyr Leu Pro 130 135 140 gct ttc gcg ctg tac ggt gtaaat ggg gcg gat gcg acg gat att cat 480 Ala Phe Ala Leu Tyr Gly Val AsnGly Ala Asp Ala Thr Asp Ile His 145 150 155 160 gac aat gcc aaa cgt ctggct gct tac ata aag aaa tca ttt taa 525 Asp Asn Ala Lys Arg Leu Ala AlaTyr Ile Lys Lys Ser Phe 165 170 2 174 PRT Bacillus amyloliquefaciens 2Val Lys Val Leu Val Leu Ala Val His Pro Asp Met Glu Asn Ser Ala 1 5 1015 Val Asn Lys Ala Trp Ala Glu Glu Leu Lys Lys His Asp Glu Leu Thr 20 2530 Val Arg Glu Leu Tyr Lys Glu Tyr Pro Asp Gly Gln Ile Asp Ala Glu 35 4045 Lys Glu Arg Gln Leu Cys Glu Gln Tyr Asp Arg Ile Val Phe Gln Phe 50 5560 Pro Leu Tyr Trp Tyr Ser Ala Pro Pro Leu Leu Lys Thr Trp Met Asp 65 7075 80 His Val Leu Ser Tyr Gly Trp Ala Tyr Gly Ser Lys Gly Lys Ala Leu 8590 95 His Gly Lys Glu Leu Met Leu Ala Val Ser Val Gly Ala Gly Glu Asp100 105 110 Ala Tyr Gln Ala Gly Gly Ser Asn His Phe Thr Leu Ser Glu LeuLeu 115 120 125 Arg Pro Phe Gln Ala Met Ala Asn Phe Thr Gly Met Thr TyrLeu Pro 130 135 140 Ala Phe Ala Leu Tyr Gly Val Asn Gly Ala Asp Ala ThrAsp Ile His 145 150 155 160 Asp Asn Ala Lys Arg Leu Ala Ala Tyr Ile LysLys Ser Phe 165 170 3 528 DNA Bacillus subtilis CDS (1)..(528) 3 atg aaaata ttg gtt ttg gca gtg cat cct cat atg gag acc tca gtt 48 Met Lys IleLeu Val Leu Ala Val His Pro His Met Glu Thr Ser Val 1 5 10 15 gtt aataag gcg tgg gct gag gaa ttg agt aaa cat gac aat atc aca 96 Val Asn LysAla Trp Ala Glu Glu Leu Ser Lys His Asp Asn Ile Thr 20 25 30 gta cgg gatctt tat aag gaa tac ccg gat gaa gcg ata gat gtt gcg 144 Val Arg Asp LeuTyr Lys Glu Tyr Pro Asp Glu Ala Ile Asp Val Ala 35 40 45 aag gaa cag cagctg tgc gag gaa tac gat cgg att gtc ttt caa ttc 192 Lys Glu Gln Gln LeuCys Glu Glu Tyr Asp Arg Ile Val Phe Gln Phe 50 55 60 ccg cta tat tgg tacagc tct ccg ccg ctc ttg aaa aaa tgg cag gat 240 Pro Leu Tyr Trp Tyr SerSer Pro Pro Leu Leu Lys Lys Trp Gln Asp 65 70 75 80 ctt gtg ctg act tatggc tgg gct ttt ggt tca gaa gga aat gcc ttg 288 Leu Val Leu Thr Tyr GlyTrp Ala Phe Gly Ser Glu Gly Asn Ala Leu 85 90 95 cat ggc aag gag ctg atgctg gct gta tca aca ggg agc gaa gca gaa 336 His Gly Lys Glu Leu Met LeuAla Val Ser Thr Gly Ser Glu Ala Glu 100 105 110 aaa tat caa gcg ggc ggagca aat cat tac tcg atc agt gag cta ttg 384 Lys Tyr Gln Ala Gly Gly AlaAsn His Tyr Ser Ile Ser Glu Leu Leu 115 120 125 aaa cca ttt cag gcc acgagt aat ctg atc ggc atg aag tat ctg cct 432 Lys Pro Phe Gln Ala Thr SerAsn Leu Ile Gly Met Lys Tyr Leu Pro 130 135 140 cca tat gtg ttc tat ggcgtg aat tat gca gct gca gag gat att tct 480 Pro Tyr Val Phe Tyr Gly ValAsn Tyr Ala Ala Ala Glu Asp Ile Ser 145 150 155 160 cac agt gca aaa cggtta gcc gaa tac atc cag cag cct ttt gtt taa 528 His Ser Ala Lys Arg LeuAla Glu Tyr Ile Gln Gln Pro Phe Val 165 170 175 4 175 PRT Bacillussubtilis 4 Met Lys Ile Leu Val Leu Ala Val His Pro His Met Glu Thr SerVal 1 5 10 15 Val Asn Lys Ala Trp Ala Glu Glu Leu Ser Lys His Asp AsnIle Thr 20 25 30 Val Arg Asp Leu Tyr Lys Glu Tyr Pro Asp Glu Ala Ile AspVal Ala 35 40 45 Lys Glu Gln Gln Leu Cys Glu Glu Tyr Asp Arg Ile Val PheGln Phe 50 55 60 Pro Leu Tyr Trp Tyr Ser Ser Pro Pro Leu Leu Lys Lys TrpGln Asp 65 70 75 80 Leu Val Leu Thr Tyr Gly Trp Ala Phe Gly Ser Glu GlyAsn Ala Leu 85 90 95 His Gly Lys Glu Leu Met Leu Ala Val Ser Thr Gly SerGlu Ala Glu 100 105 110 Lys Tyr Gln Ala Gly Gly Ala Asn His Tyr Ser IleSer Glu Leu Leu 115 120 125 Lys Pro Phe Gln Ala Thr Ser Asn Leu Ile GlyMet Lys Tyr Leu Pro 130 135 140 Pro Tyr Val Phe Tyr Gly Val Asn Tyr AlaAla Ala Glu Asp Ile Ser 145 150 155 160 His Ser Ala Lys Arg Leu Ala GluTyr Ile Gln Gln Pro Phe Val 165 170 175 5 525 DNA Bacillus subtilis CDS(1)..(525) 5 atg aaa aca tta gtt atc gtt ata cat cct aat ttg gaa acg tctgtt 48 Met Lys Thr Leu Val Ile Val Ile His Pro Asn Leu Glu Thr Ser Val 15 10 15 gtc aac aaa acc tgg atg aat cgt tta aag caa gag aaa gac att acg96 Val Asn Lys Thr Trp Met Asn Arg Leu Lys Gln Glu Lys Asp Ile Thr 20 2530 gtt cat gac ctg tac ggt gaa tac cct aat ttt atc att gat gta gaa 144Val His Asp Leu Tyr Gly Glu Tyr Pro Asn Phe Ile Ile Asp Val Glu 35 40 45aaa gag cag cag ctc ctg tta gat cat gag cgt atc gtt ttt cag ttc 192 LysGlu Gln Gln Leu Leu Leu Asp His Glu Arg Ile Val Phe Gln Phe 50 55 60 ccaatg tat tgg tac agc agt ccc gcg tta ctc aaa caa tgg gaa gat 240 Pro MetTyr Trp Tyr Ser Ser Pro Ala Leu Leu Lys Gln Trp Glu Asp 65 70 75 80 gatgtg tta aca cat ggc tgg gct tat gga act gga gga act aaa ttg 288 Asp ValLeu Thr His Gly Trp Ala Tyr Gly Thr Gly Gly Thr Lys Leu 85 90 95 cat ggaaaa gaa cta ctc tta gct atc tcc tca ggc gca cag gaa tct 336 His Gly LysGlu Leu Leu Leu Ala Ile Ser Ser Gly Ala Gln Glu Ser 100 105 110 gat tatcaa gca ggc gga gaa tat aat atc acg atc agc gag ctt atc 384 Asp Tyr GlnAla Gly Gly Glu Tyr Asn Ile Thr Ile Ser Glu Leu Ile 115 120 125 aga ccgttt caa gtc act gct aac tat ata gga atg cgt ttt ctt cct 432 Arg Pro PheGln Val Thr Ala Asn Tyr Ile Gly Met Arg Phe Leu Pro 130 135 140 gcg tttaca caa tat ggg aca ctt cat ctt tca aaa gaa gat gtt aag 480 Ala Phe ThrGln Tyr Gly Thr Leu His Leu Ser Lys Glu Asp Val Lys 145 150 155 160 aacagt gcg gag aga ttg gtt gac tat ctt aaa gcc gag cat taa 525 Asn Ser AlaGlu Arg Leu Val Asp Tyr Leu Lys Ala Glu His 165 170 6 174 PRT Bacillussubtilis 6 Met Lys Thr Leu Val Ile Val Ile His Pro Asn Leu Glu Thr SerVal 1 5 10 15 Val Asn Lys Thr Trp Met Asn Arg Leu Lys Gln Glu Lys AspIle Thr 20 25 30 Val His Asp Leu Tyr Gly Glu Tyr Pro Asn Phe Ile Ile AspVal Glu 35 40 45 Lys Glu Gln Gln Leu Leu Leu Asp His Glu Arg Ile Val PheGln Phe 50 55 60 Pro Met Tyr Trp Tyr Ser Ser Pro Ala Leu Leu Lys Gln TrpGlu Asp 65 70 75 80 Asp Val Leu Thr His Gly Trp Ala Tyr Gly Thr Gly GlyThr Lys Leu 85 90 95 His Gly Lys Glu Leu Leu Leu Ala Ile Ser Ser Gly AlaGln Glu Ser 100 105 110 Asp Tyr Gln Ala Gly Gly Glu Tyr Asn Ile Thr IleSer Glu Leu Ile 115 120 125 Arg Pro Phe Gln Val Thr Ala Asn Tyr Ile GlyMet Arg Phe Leu Pro 130 135 140 Ala Phe Thr Gln Tyr Gly Thr Leu His LeuSer Lys Glu Asp Val Lys 145 150 155 160 Asn Ser Ala Glu Arg Leu Val AspTyr Leu Lys Ala Glu His 165 170 7 594 DNA Bacillus subtilis CDS(1)..(594) 7 atg gat cat atg aaa aca ctc gta ctc gtt gta cat ccg aat atagaa 48 Met Asp His Met Lys Thr Leu Val Leu Val Val His Pro Asn Ile Glu 15 10 15 tcc tct cgt atc aat aaa aag tgg aaa gaa gcc gtt tta agt gaa cca96 Ser Ser Arg Ile Asn Lys Lys Trp Lys Glu Ala Val Leu Ser Glu Pro 20 2530 gat gta act gtc cat gat ctt tat gaa aaa tat cgc gat caa cca att 144Asp Val Thr Val His Asp Leu Tyr Glu Lys Tyr Arg Asp Gln Pro Ile 35 40 45gat gtg gaa ttt gaa caa cag cag ctc ctg gcc cat gac cgt atc gtt 192 AspVal Glu Phe Glu Gln Gln Gln Leu Leu Ala His Asp Arg Ile Val 50 55 60 tttcag ttt cca tta tac tgg tac agc agc cca ccg ctt tta aaa cag 240 Phe GlnPhe Pro Leu Tyr Trp Tyr Ser Ser Pro Pro Leu Leu Lys Gln 65 70 75 80 tggttt gat gaa gtg ttt acg ttt ggc tgg gct cat ggt ccc ggc gga 288 Trp PheAsp Glu Val Phe Thr Phe Gly Trp Ala His Gly Pro Gly Gly 85 90 95 aat aaattg aag ggg aaa gag tgg gta act gcc atg tcc atc ggt tca 336 Asn Lys LeuLys Gly Lys Glu Trp Val Thr Ala Met Ser Ile Gly Ser 100 105 110 cct gaacac tct tat caa gcc ggc gga tat aac ttg ttt tcg ata agc 384 Pro Glu HisSer Tyr Gln Ala Gly Gly Tyr Asn Leu Phe Ser Ile Ser 115 120 125 gag ctgaca aaa ccg ttc caa gca tct gcc cat tta gta ggc atg acc 432 Glu Leu ThrLys Pro Phe Gln Ala Ser Ala His Leu Val Gly Met Thr 130 135 140 tat ctgcct tcc ttt gcc gaa tat cgc gcc aat aca atc agt gac caa 480 Tyr Leu ProSer Phe Ala Glu Tyr Arg Ala Asn Thr Ile Ser Asp Gln 145 150 155 160 gaaatt gcc gaa agt gcg aat cgg tat gta aag cat att aca aat ata 528 Glu IleAla Glu Ser Ala Asn Arg Tyr Val Lys His Ile Thr Asn Ile 165 170 175 gaatta aac ccg aag gtt cgc ctg caa agg tat ttg aaa cag ctg gag 576 Glu LeuAsn Pro Lys Val Arg Leu Gln Arg Tyr Leu Lys Gln Leu Glu 180 185 190 agtgtc gat tta aca taa 594 Ser Val Asp Leu Thr 195 8 197 PRT Bacillussubtilis 8 Met Asp His Met Lys Thr Leu Val Leu Val Val His Pro Asn IleGlu 1 5 10 15 Ser Ser Arg Ile Asn Lys Lys Trp Lys Glu Ala Val Leu SerGlu Pro 20 25 30 Asp Val Thr Val His Asp Leu Tyr Glu Lys Tyr Arg Asp GlnPro Ile 35 40 45 Asp Val Glu Phe Glu Gln Gln Gln Leu Leu Ala His Asp ArgIle Val 50 55 60 Phe Gln Phe Pro Leu Tyr Trp Tyr Ser Ser Pro Pro Leu LeuLys Gln 65 70 75 80 Trp Phe Asp Glu Val Phe Thr Phe Gly Trp Ala His GlyPro Gly Gly 85 90 95 Asn Lys Leu Lys Gly Lys Glu Trp Val Thr Ala Met SerIle Gly Ser 100 105 110 Pro Glu His Ser Tyr Gln Ala Gly Gly Tyr Asn LeuPhe Ser Ile Ser 115 120 125 Glu Leu Thr Lys Pro Phe Gln Ala Ser Ala HisLeu Val Gly Met Thr 130 135 140 Tyr Leu Pro Ser Phe Ala Glu Tyr Arg AlaAsn Thr Ile Ser Asp Gln 145 150 155 160 Glu Ile Ala Glu Ser Ala Asn ArgTyr Val Lys His Ile Thr Asn Ile 165 170 175 Glu Leu Asn Pro Lys Val ArgLeu Gln Arg Tyr Leu Lys Gln Leu Glu 180 185 190 Ser Val Asp Leu Thr 1959 630 DNA Bacillus subtilis CDS (1)..(630) 9 atg atc aaa aca aac gat tttatg gaa att atg aaa ggc cgc cgt tct 48 Met Ile Lys Thr Asn Asp Phe MetGlu Ile Met Lys Gly Arg Arg Ser 1 5 10 15 atc cgc aac tat gat ccg gcagta aaa atc agc aaa gaa gaa atg aca 96 Ile Arg Asn Tyr Asp Pro Ala ValLys Ile Ser Lys Glu Glu Met Thr 20 25 30 gag atc tta gag gaa gca aca actgcc cca tct tct gtt aac gcg cag 144 Glu Ile Leu Glu Glu Ala Thr Thr AlaPro Ser Ser Val Asn Ala Gln 35 40 45 cca tgg cgt ttt ctt gtc att gac agcccg gaa gga aaa gaa aag ctc 192 Pro Trp Arg Phe Leu Val Ile Asp Ser ProGlu Gly Lys Glu Lys Leu 50 55 60 gca ccg ctt gca agc ttt aac caa aca caagtc aca aca tca tct gct 240 Ala Pro Leu Ala Ser Phe Asn Gln Thr Gln ValThr Thr Ser Ser Ala 65 70 75 80 gtc atc gct gta ttt gca gac atg aac aacgca gac tat cta gaa gaa 288 Val Ile Ala Val Phe Ala Asp Met Asn Asn AlaAsp Tyr Leu Glu Glu 85 90 95 atc tat tca aaa gcc gtg gaa ctt ggt tac atgccg cag gag gtc aaa 336 Ile Tyr Ser Lys Ala Val Glu Leu Gly Tyr Met ProGln Glu Val Lys 100 105 110 gac aga caa atc gcc gcg ctg acc gca cat tttgaa aag ctt ccg gca 384 Asp Arg Gln Ile Ala Ala Leu Thr Ala His Phe GluLys Leu Pro Ala 115 120 125 cag gtc aac cgt gaa acg atc ctg att gac ggaggt ctt gtt tcc atg 432 Gln Val Asn Arg Glu Thr Ile Leu Ile Asp Gly GlyLeu Val Ser Met 130 135 140 cag ctg atg ctg act gca cgc gcg cat ggc tacgat aca aac ccg atc 480 Gln Leu Met Leu Thr Ala Arg Ala His Gly Tyr AspThr Asn Pro Ile 145 150 155 160 ggc gga tac gat aaa gaa aac atc gcg gaaacc ttc gga tta gat aaa 528 Gly Gly Tyr Asp Lys Glu Asn Ile Ala Glu ThrPhe Gly Leu Asp Lys 165 170 175 gaa cgt tat gta ccg gtt atg cta ctt tctatc gga aaa gca gca gac 576 Glu Arg Tyr Val Pro Val Met Leu Leu Ser IleGly Lys Ala Ala Asp 180 185 190 gaa ggc tat gct tcc tac cgt ctg ccg attgat aca att gca gaa tgg 624 Glu Gly Tyr Ala Ser Tyr Arg Leu Pro Ile AspThr Ile Ala Glu Trp 195 200 205 aaa taa 630 Lys 10 209 PRT Bacillussubtilis 10 Met Ile Lys Thr Asn Asp Phe Met Glu Ile Met Lys Gly Arg ArgSer 1 5 10 15 Ile Arg Asn Tyr Asp Pro Ala Val Lys Ile Ser Lys Glu GluMet Thr 20 25 30 Glu Ile Leu Glu Glu Ala Thr Thr Ala Pro Ser Ser Val AsnAla Gln 35 40 45 Pro Trp Arg Phe Leu Val Ile Asp Ser Pro Glu Gly Lys GluLys Leu 50 55 60 Ala Pro Leu Ala Ser Phe Asn Gln Thr Gln Val Thr Thr SerSer Ala 65 70 75 80 Val Ile Ala Val Phe Ala Asp Met Asn Asn Ala Asp TyrLeu Glu Glu 85 90 95 Ile Tyr Ser Lys Ala Val Glu Leu Gly Tyr Met Pro GlnGlu Val Lys 100 105 110 Asp Arg Gln Ile Ala Ala Leu Thr Ala His Phe GluLys Leu Pro Ala 115 120 125 Gln Val Asn Arg Glu Thr Ile Leu Ile Asp GlyGly Leu Val Ser Met 130 135 140 Gln Leu Met Leu Thr Ala Arg Ala His GlyTyr Asp Thr Asn Pro Ile 145 150 155 160 Gly Gly Tyr Asp Lys Glu Asn IleAla Glu Thr Phe Gly Leu Asp Lys 165 170 175 Glu Arg Tyr Val Pro Val MetLeu Leu Ser Ile Gly Lys Ala Ala Asp 180 185 190 Glu Gly Tyr Ala Ser TyrArg Leu Pro Ile Asp Thr Ile Ala Glu Trp 195 200 205 Lys 11 609 DNABacillus subtilis CDS (1)..(609) 11 atg acg aat act ctg gat gtt tta aaagca cgt gca tct gta aag gaa 48 Met Thr Asn Thr Leu Asp Val Leu Lys AlaArg Ala Ser Val Lys Glu 1 5 10 15 tat gat aca aat gcc ccg atc tct aaggag gag ctg act gag cta tta 96 Tyr Asp Thr Asn Ala Pro Ile Ser Lys GluGlu Leu Thr Glu Leu Leu 20 25 30 gac ctt gcc act aaa gcg cct tct gct tggaac ctt cag cat tgg cat 144 Asp Leu Ala Thr Lys Ala Pro Ser Ala Trp AsnLeu Gln His Trp His 35 40 45 ttt aca gta ttc cac agc gat gaa tca aaa gcggag ctt ctt cct gta 192 Phe Thr Val Phe His Ser Asp Glu Ser Lys Ala GluLeu Leu Pro Val 50 55 60 gcg tat aat caa aaa caa atc gtt gag tct tct gctgtt gtt gcc att 240 Ala Tyr Asn Gln Lys Gln Ile Val Glu Ser Ser Ala ValVal Ala Ile 65 70 75 80 tta ggc gat tta aag gca aat gaa aac ggt gaa gaagtt tat gct gaa 288 Leu Gly Asp Leu Lys Ala Asn Glu Asn Gly Glu Glu ValTyr Ala Glu 85 90 95 tta gca agc caa ggc tat att acg gat gaa atc aaa caaaca ttg ctc 336 Leu Ala Ser Gln Gly Tyr Ile Thr Asp Glu Ile Lys Gln ThrLeu Leu 100 105 110 ggc caa atc aac ggt gct tac caa agc gag caa ttc gcacgt gat tcc 384 Gly Gln Ile Asn Gly Ala Tyr Gln Ser Glu Gln Phe Ala ArgAsp Ser 115 120 125 gct ttc tta aat gct tct tta gct gct atg cag ctt atgatt gcc gca 432 Ala Phe Leu Asn Ala Ser Leu Ala Ala Met Gln Leu Met IleAla Ala 130 135 140 aaa gca aaa ggt tat gac act tgc gca atc ggc gga tttaac aaa gag 480 Lys Ala Lys Gly Tyr Asp Thr Cys Ala Ile Gly Gly Phe AsnLys Glu 145 150 155 160 cag ttc caa aag caa ttt gat atc agt gag cgc tatgtt ccg gtt atg 528 Gln Phe Gln Lys Gln Phe Asp Ile Ser Glu Arg Tyr ValPro Val Met 165 170 175 ctt att tca atc ggc aaa gca gtg aag cct gcg catcaa agc aac cgt 576 Leu Ile Ser Ile Gly Lys Ala Val Lys Pro Ala His GlnSer Asn Arg 180 185 190 ctg ccg ctt tca aaa gta tca act tgg ctg taa 609Leu Pro Leu Ser Lys Val Ser Thr Trp Leu 195 200 12 202 PRT Bacillussubtilis 12 Met Thr Asn Thr Leu Asp Val Leu Lys Ala Arg Ala Ser Val LysGlu 1 5 10 15 Tyr Asp Thr Asn Ala Pro Ile Ser Lys Glu Glu Leu Thr GluLeu Leu 20 25 30 Asp Leu Ala Thr Lys Ala Pro Ser Ala Trp Asn Leu Gln HisTrp His 35 40 45 Phe Thr Val Phe His Ser Asp Glu Ser Lys Ala Glu Leu LeuPro Val 50 55 60 Ala Tyr Asn Gln Lys Gln Ile Val Glu Ser Ser Ala Val ValAla Ile 65 70 75 80 Leu Gly Asp Leu Lys Ala Asn Glu Asn Gly Glu Glu ValTyr Ala Glu 85 90 95 Leu Ala Ser Gln Gly Tyr Ile Thr Asp Glu Ile Lys GlnThr Leu Leu 100 105 110 Gly Gln Ile Asn Gly Ala Tyr Gln Ser Glu Gln PheAla Arg Asp Ser 115 120 125 Ala Phe Leu Asn Ala Ser Leu Ala Ala Met GlnLeu Met Ile Ala Ala 130 135 140 Lys Ala Lys Gly Tyr Asp Thr Cys Ala IleGly Gly Phe Asn Lys Glu 145 150 155 160 Gln Phe Gln Lys Gln Phe Asp IleSer Glu Arg Tyr Val Pro Val Met 165 170 175 Leu Ile Ser Ile Gly Lys AlaVal Lys Pro Ala His Gln Ser Asn Arg 180 185 190 Leu Pro Leu Ser Lys ValSer Thr Trp Leu 195 200 13 555 DNA Escherichia coli CDS (1)..(555) 13atg atg tct cag cca gcg aaa gtt ttg ctg ctg tat gcc cat ccg gaa 48 MetMet Ser Gln Pro Ala Lys Val Leu Leu Leu Tyr Ala His Pro Glu 1 5 10 15tct cag gac tcg gtg gca aac cgg gta ctg ctt aaa ccg gcc acg cag 96 SerGln Asp Ser Val Ala Asn Arg Val Leu Leu Lys Pro Ala Thr Gln 20 25 30 ctcagc aat gtt acc gtg cac gac ctt tac gcg cac tat ccc gat ttt 144 Leu SerAsn Val Thr Val His Asp Leu Tyr Ala His Tyr Pro Asp Phe 35 40 45 ttt attgat atc ccc cgt gag cag gca tta ctg cgc gag cac gag gtg 192 Phe Ile AspIle Pro Arg Glu Gln Ala Leu Leu Arg Glu His Glu Val 50 55 60 att gtc tttcag cat cct ctt tat acc tat agc tgc ccg gcg cta ctg 240 Ile Val Phe GlnHis Pro Leu Tyr Thr Tyr Ser Cys Pro Ala Leu Leu 65 70 75 80 aaa gag tggctg gac cgg gta tta agt cgt ggt ttt gcc agc ggg ccg 288 Lys Glu Trp LeuAsp Arg Val Leu Ser Arg Gly Phe Ala Ser Gly Pro 85 90 95 gga gga aac caactg gcg gga aag tac tgg cgt agc gtg att acc acc 336 Gly Gly Asn Gln LeuAla Gly Lys Tyr Trp Arg Ser Val Ile Thr Thr 100 105 110 ggc gag ccg gaaagt gct tac cgt tat gac gcg ctg aat cgc tac ccg 384 Gly Glu Pro Glu SerAla Tyr Arg Tyr Asp Ala Leu Asn Arg Tyr Pro 115 120 125 atg agc gat gtgctg cgc ccc ttt gaa ctg gcg gcg ggc atg tgc cgg 432 Met Ser Asp Val LeuArg Pro Phe Glu Leu Ala Ala Gly Met Cys Arg 130 135 140 atg cat tgg ttaagt ccc atc att att tac tgg gcg aga cgg caa agc 480 Met His Trp Leu SerPro Ile Ile Ile Tyr Trp Ala Arg Arg Gln Ser 145 150 155 160 gca cag gagctg gcg agc cac gcc aga gcc tac ggt gac tgg ctg gca 528 Ala Gln Glu LeuAla Ser His Ala Arg Ala Tyr Gly Asp Trp Leu Ala 165 170 175 aat ccg ctgtct cca gga ggc cgc tga 555 Asn Pro Leu Ser Pro Gly Gly Arg 180 14 184PRT Escherichia coli 14 Met Met Ser Gln Pro Ala Lys Val Leu Leu Leu TyrAla His Pro Glu 1 5 10 15 Ser Gln Asp Ser Val Ala Asn Arg Val Leu LeuLys Pro Ala Thr Gln 20 25 30 Leu Ser Asn Val Thr Val His Asp Leu Tyr AlaHis Tyr Pro Asp Phe 35 40 45 Phe Ile Asp Ile Pro Arg Glu Gln Ala Leu LeuArg Glu His Glu Val 50 55 60 Ile Val Phe Gln His Pro Leu Tyr Thr Tyr SerCys Pro Ala Leu Leu 65 70 75 80 Lys Glu Trp Leu Asp Arg Val Leu Ser ArgGly Phe Ala Ser Gly Pro 85 90 95 Gly Gly Asn Gln Leu Ala Gly Lys Tyr TrpArg Ser Val Ile Thr Thr 100 105 110 Gly Glu Pro Glu Ser Ala Tyr Arg TyrAsp Ala Leu Asn Arg Tyr Pro 115 120 125 Met Ser Asp Val Leu Arg Pro PheGlu Leu Ala Ala Gly Met Cys Arg 130 135 140 Met His Trp Leu Ser Pro IleIle Ile Tyr Trp Ala Arg Arg Gln Ser 145 150 155 160 Ala Gln Glu Leu AlaSer His Ala Arg Ala Tyr Gly Asp Trp Leu Ala 165 170 175 Asn Pro Leu SerPro Gly Gly Arg 180 15 531 DNA Escherichia coli CDS (1)..(531) 15 atgatt ctt ata att tat gcg cat ccg tat ccg cat cat tcc cat gcg 48 Met IleLeu Ile Ile Tyr Ala His Pro Tyr Pro His His Ser His Ala 1 5 10 15 aataaa cgg atg ctt gaa cag gca agg acg ctg gaa ggc gtc gaa att 96 Asn LysArg Met Leu Glu Gln Ala Arg Thr Leu Glu Gly Val Glu Ile 20 25 30 cgc tctctt tat caa ctc tat cct gac ttc aat atc gat att gcc gcc 144 Arg Ser LeuTyr Gln Leu Tyr Pro Asp Phe Asn Ile Asp Ile Ala Ala 35 40 45 gag cag gaggcg ctg tct cgc gcc gat ctg atc gtc tgg cag cat ccg 192 Glu Gln Glu AlaLeu Ser Arg Ala Asp Leu Ile Val Trp Gln His Pro 50 55 60 atg cag tgg tacagc att cct ccg ctc ctc aaa ctt tgg atc gat aaa 240 Met Gln Trp Tyr SerIle Pro Pro Leu Leu Lys Leu Trp Ile Asp Lys 65 70 75 80 gtt ttc tcc cacggc tgg gct tac ggt cat ggc ggc acg gcg ctg cat 288 Val Phe Ser His GlyTrp Ala Tyr Gly His Gly Gly Thr Ala Leu His 85 90 95 ggc aaa cat ttg ctgtgg gcg gtg acg acc ggc ggc ggg gaa agc cat 336 Gly Lys His Leu Leu TrpAla Val Thr Thr Gly Gly Gly Glu Ser His 100 105 110 ttt gaa att ggt gcgcat ccg ggc ttt gat gtg ctg tcg cag ccg cta 384 Phe Glu Ile Gly Ala HisPro Gly Phe Asp Val Leu Ser Gln Pro Leu 115 120 125 cag gcg acg gca atctac tgc ggg ctg aac tgg ctg cca ccg ttt gcc 432 Gln Ala Thr Ala Ile TyrCys Gly Leu Asn Trp Leu Pro Pro Phe Ala 130 135 140 atg cac tgc acc tttatt tgt gac gac gaa acc ctc gaa ggg cag gcg 480 Met His Cys Thr Phe IleCys Asp Asp Glu Thr Leu Glu Gly Gln Ala 145 150 155 160 cgt cac tat aagcaa cgt ctg ctg gaa tgg cag gag gcc cat cat gga 528 Arg His Tyr Lys GlnArg Leu Leu Glu Trp Gln Glu Ala His His Gly 165 170 175 tag 531 16 176PRT Escherichia coli 16 Met Ile Leu Ile Ile Tyr Ala His Pro Tyr Pro HisHis Ser His Ala 1 5 10 15 Asn Lys Arg Met Leu Glu Gln Ala Arg Thr LeuGlu Gly Val Glu Ile 20 25 30 Arg Ser Leu Tyr Gln Leu Tyr Pro Asp Phe AsnIle Asp Ile Ala Ala 35 40 45 Glu Gln Glu Ala Leu Ser Arg Ala Asp Leu IleVal Trp Gln His Pro 50 55 60 Met Gln Trp Tyr Ser Ile Pro Pro Leu Leu LysLeu Trp Ile Asp Lys 65 70 75 80 Val Phe Ser His Gly Trp Ala Tyr Gly HisGly Gly Thr Ala Leu His 85 90 95 Gly Lys His Leu Leu Trp Ala Val Thr ThrGly Gly Gly Glu Ser His 100 105 110 Phe Glu Ile Gly Ala His Pro Gly PheAsp Val Leu Ser Gln Pro Leu 115 120 125 Gln Ala Thr Ala Ile Tyr Cys GlyLeu Asn Trp Leu Pro Pro Phe Ala 130 135 140 Met His Cys Thr Phe Ile CysAsp Asp Glu Thr Leu Glu Gly Gln Ala 145 150 155 160 Arg His Tyr Lys GlnArg Leu Leu Glu Trp Gln Glu Ala His His 165 170 175 Gly 17 220 PRTHaemophilus influenzae 17 Met Thr Gln Leu Thr Arg Glu Gln Val Leu GluLeu Phe His Gln Arg 1 5 10 15 Ser Ser Thr Arg Tyr Tyr Asp Pro Thr LysLys Ile Ser Asp Glu Asp 20 25 30 Phe Glu Cys Ile Leu Glu Cys Gly Arg LeuSer Pro Ser Ser Val Gly 35 40 45 Ser Glu Pro Trp Lys Phe Leu Val Ile GlnAsn Lys Thr Leu Arg Glu 50 55 60 Lys Met Lys Pro Phe Ser Trp Gly Met IleAsn Gln Leu Asp Asn Cys 65 70 75 80 Ser His Leu Val Val Ile Leu Ala LysLys Asn Ala Arg Tyr Asp Ser 85 90 95 Pro Phe Phe Val Asp Val Met Ala ArgLys Gly Leu Asn Ala Glu Gln 100 105 110 Gln Gln Ala Ala Leu Thr Lys TyrLys Ala Leu Gln Glu Glu Asp Met 115 120 125 Lys Leu Leu Glu Asn Asp ArgThr Leu Phe Asp Trp Cys Ser Lys Gln 130 135 140 Thr Tyr Ile Ala Leu AlaAsn Met Leu Thr Gly Ala Ser Ala Leu Gly 145 150 155 160 Ile Asp Ser CysPro Ile Glu Gly Phe His Tyr Asp Lys Met Asn Glu 165 170 175 Cys Leu AlaGlu Glu Gly Leu Phe Asp Pro Gln Glu Tyr Ala Val Ser 180 185 190 Val AlaAla Thr Phe Gly Tyr Arg Ser Arg Asp Ile Ala Lys Lys Ser 195 200 205 ArgLys Gly Leu Asp Glu Val Val Lys Trp Val Gly 210 215 220 18 205 PRTThermus aquaticus 18 Met Glu Ala Thr Leu Pro Val Leu Asp Ala Lys Thr AlaAla Leu Lys 1 5 10 15 Arg Arg Ser Ile Arg Arg Tyr Arg Lys Asp Pro ValPro Glu Gly Leu 20 25 30 Leu Arg Glu Ile Leu Glu Ala Ala Leu Arg Ala ProSer Ala Trp Asn 35 40 45 Leu Gln Pro Trp Arg Ile Val Val Val Arg Asp ProAla Thr Lys Arg 50 55 60 Ala Leu Arg Glu Ala Ala Phe Gly Gln Ala His ValGlu Glu Ala Pro 65 70 75 80 Val Val Leu Val Leu Tyr Ala Asp Leu Glu AspAla Leu Ala His Leu 85 90 95 Asp Glu Val Ile His Pro Gly Val Gln Gly GluArg Arg Glu Ala Gln 100 105 110 Lys Gln Ala Ile Gln Arg Ala Phe Ala AlaMet Gly Gln Glu Ala Arg 115 120 125 Lys Ala Trp Ala Ser Gly Gln Ser TyrIle Leu Leu Gly Tyr Leu Leu 130 135 140 Leu Leu Leu Glu Ala Tyr Gly LeuGly Ser Val Pro Met Leu Gly Phe 145 150 155 160 Asp Pro Glu Arg Val ArgAla Ile Leu Gly Leu Pro Ser Arg Ala Ala 165 170 175 Ile Pro Ala Leu ValAla Leu Gly Tyr Pro Ala Glu Glu Gly Tyr Pro 180 185 190 Ser His Arg LeuPro Leu Glu Arg Val Val Leu Trp Arg 195 200 205 19 200 PRT SynechocystisPCC6803 19 Met Asp Thr Phe Asp Ala Ile Tyr Gln Arg Arg Ser Val Lys HisPhe 1 5 10 15 Asp Pro Asp His Arg Leu Thr Ala Glu Glu Glu Arg Lys LeuHis Glu 20 25 30 Ala Ala Ile Gln Ala Pro Thr Ser Phe Asn Ile Gln Leu TrpArg Phe 35 40 45 Leu Ile Ile Arg Asp Pro Gln Leu Arg Gln Thr Ile Arg GluLys Tyr 50 55 60 Gly Asn Gln Ala Gln Met Thr Asp Ala Ser Leu Leu Ile LeuVal Ala 65 70 75 80 Ala Asp Val Asn Ala Trp Asp Lys Asp Pro Ala Arg TyrTrp Arg Asn 85 90 95 Ala Pro Arg Glu Val Ala Asn Tyr Leu Val Gly Ala IleAla Phe Tyr 100 105 110 Gly Gly Lys Pro Gln Leu Gln Arg Asp Glu Ala GlnArg Ser Ile Gly 115 120 125 Met Ala Met Gln Asn Leu Met Leu Ala Ala LysAla Met Gly Tyr Asp 130 135 140 Ser Cys Pro Met Ile Gly Phe Asp Leu GlnLys Val Ala Glu Leu Val 145 150 155 160 Lys Leu Pro Ala Asp Tyr Ala IleGly Pro Met Val Ala Ile Gly Lys 165 170 175 Arg Thr Glu Asp Ala Arg AlaLys Gly Gly Gln Thr Pro Leu Glu Glu 180 185 190 Leu Val Trp Glu Asn SerPhe Ala 195 200 20 172 PRT Archaeoglobus fulgidus 20 Met Glu Cys Leu AspLeu Leu Phe Arg Arg Val Ser Ile Arg Lys Phe 1 5 10 15 Thr Gln Asp AspVal Asp Asp Glu Ile Leu Met Lys Ile Leu Glu Ala 20 25 30 Gly Asn Ala AlaPro Ser Ala Gly Asn Leu Gln Ala Arg Asp Phe Val 35 40 45 Val Ile Arg AsnPro Glu Thr Lys Lys Arg Leu Ala Met Ala Ala Leu 50 55 60 Lys Gln Met PheIle Ala Glu Ala Pro Val Val Ile Val Val Cys Ala 65 70 75 80 Asn Tyr ProArg Ser Met Arg Val Tyr Gly Glu Arg Gly Arg Leu Tyr 85 90 95 Ala Glu GlnAsp Ala Thr Ala Ala Ile Glu Asn Ile Leu Leu Ala Val 100 105 110 Thr AlaLeu Asn Leu Gly Ala Val Trp Val Gly Ala Phe Asp Glu Glu 115 120 125 GlnVal Ser Glu Ile Leu Glu Leu Pro Glu Tyr Val Arg Pro Met Ala 130 135 140Ile Ile Pro Ile Gly His Pro Ala Glu Asn Pro Ser Pro Arg Asn Arg 145 150155 160 Tyr Pro Val Ser Met Leu Thr His Phe Glu Lys Trp 165 170 21 174PRT Archaeoglobus fulgidus 21 Met Glu Glu Cys Leu Lys Met Ile Tyr ThrArg Arg Ser Ile Arg Val 1 5 10 15 Tyr Ser Asp Arg Gln Ile Ser Asp GluAsp Ile Glu Lys Ile Leu Lys 20 25 30 Ala Ala Met Leu Ala Pro Ser Ala GlyAsn Glu Gln Pro Trp His Phe 35 40 45 Ile Val Val Arg Asp Arg Glu Met LeuLys Lys Met Ser Glu Ala Phe 50 55 60 Thr Phe Gly Gln Met Leu Pro Asn AlaSer Ala Ala Ile Val Val Cys 65 70 75 80 Ala Asp Pro Lys Leu Ser Lys TyrPro Tyr Asp Met Trp Val Gln Asp 85 90 95 Cys Ser Ala Ala Thr Glu Asn IleLeu Leu Ala Ala Arg Cys Leu Gly 100 105 110 Ile Gly Ser Val Trp Leu GlyVal Tyr Pro Arg Glu Glu Arg Met Lys 115 120 125 Ala Leu Arg Glu Leu LeuGly Ile Pro Glu Asn Ile Val Val Phe Ser 130 135 140 Val Val Ser Leu GlyTyr Pro Lys Asp Glu Lys Asp Phe Tyr Glu Ala 145 150 155 160 Asp Asp ArgPhe Asn Pro Asp Arg Ile His Arg Glu Lys Trp 165 170 22 606 DNACampylobacter jejuni CDS (1)..(606) 22 atg aaa aaa gaa ctt gaa att tttagc aca aga tat tct tgt aga aat 48 Met Lys Lys Glu Leu Glu Ile Phe SerThr Arg Tyr Ser Cys Arg Asn 1 5 10 15 ttt aaa aat gaa aaa ctc aaa aaagag gat tta aat tct att tta gaa 96 Phe Lys Asn Glu Lys Leu Lys Lys GluAsp Leu Asn Ser Ile Leu Glu 20 25 30 ata gca aga tta agc ccc agt tcc ttggga ctg gaa cct tgg aaa ttt 144 Ile Ala Arg Leu Ser Pro Ser Ser Leu GlyLeu Glu Pro Trp Lys Phe 35 40 45 ata gta gtg caa gat gag aaa aga aaa gaagaa ctt tct aaa att tgc 192 Ile Val Val Gln Asp Glu Lys Arg Lys Glu GluLeu Ser Lys Ile Cys 50 55 60 aat caa caa aaa cat gta aaa gat tgt gct gcatta att ata atc att 240 Asn Gln Gln Lys His Val Lys Asp Cys Ala Ala LeuIle Ile Ile Ile 65 70 75 80 tca aga ctt gat ttt ttg gat tat ttt gaa gaaaaa ctt aga aaa aga 288 Ser Arg Leu Asp Phe Leu Asp Tyr Phe Glu Glu LysLeu Arg Lys Arg 85 90 95 gat atg agt gaa aca gaa atg caa aaa cgc tta gatact tat atg cct 336 Asp Met Ser Glu Thr Glu Met Gln Lys Arg Leu Asp ThrTyr Met Pro 100 105 110 ttt tta aaa tct cta aat caa gaa caa aaa ata tcttat gca aga gaa 384 Phe Leu Lys Ser Leu Asn Gln Glu Gln Lys Ile Ser TyrAla Arg Glu 115 120 125 caa gct cat ata gct cta gct agc ata ctt tac agtgct aat gct tta 432 Gln Ala His Ile Ala Leu Ala Ser Ile Leu Tyr Ser AlaAsn Ala Leu 130 135 140 aat ata gca agc tgc act ata ggt ggt ttt gat aaagaa aag ctt gat 480 Asn Ile Ala Ser Cys Thr Ile Gly Gly Phe Asp Lys GluLys Leu Asp 145 150 155 160 tct tat tta tca ctt gat att caa aaa gaa agatca agt ttg gtg gtg 528 Ser Tyr Leu Ser Leu Asp Ile Gln Lys Glu Arg SerSer Leu Val Val 165 170 175 gct tta gga tat tgc aac gat aaa aaa aat cctcaa aaa aat cgt ttt 576 Ala Leu Gly Tyr Cys Asn Asp Lys Lys Asn Pro GlnLys Asn Arg Phe 180 185 190 agt ttt gat gaa gtt gta aaa ttt att taa 606Ser Phe Asp Glu Val Val Lys Phe Ile 195 200 23 201 PRT Campylobacterjejuni 23 Met Lys Lys Glu Leu Glu Ile Phe Ser Thr Arg Tyr Ser Cys ArgAsn 1 5 10 15 Phe Lys Asn Glu Lys Leu Lys Lys Glu Asp Leu Asn Ser IleLeu Glu 20 25 30 Ile Ala Arg Leu Ser Pro Ser Ser Leu Gly Leu Glu Pro TrpLys Phe 35 40 45 Ile Val Val Gln Asp Glu Lys Arg Lys Glu Glu Leu Ser LysIle Cys 50 55 60 Asn Gln Gln Lys His Val Lys Asp Cys Ala Ala Leu Ile IleIle Ile 65 70 75 80 Ser Arg Leu Asp Phe Leu Asp Tyr Phe Glu Glu Lys LeuArg Lys Arg 85 90 95 Asp Met Ser Glu Thr Glu Met Gln Lys Arg Leu Asp ThrTyr Met Pro 100 105 110 Phe Leu Lys Ser Leu Asn Gln Glu Gln Lys Ile SerTyr Ala Arg Glu 115 120 125 Gln Ala His Ile Ala Leu Ala Ser Ile Leu TyrSer Ala Asn Ala Leu 130 135 140 Asn Ile Ala Ser Cys Thr Ile Gly Gly PheAsp Lys Glu Lys Leu Asp 145 150 155 160 Ser Tyr Leu Ser Leu Asp Ile GlnLys Glu Arg Ser Ser Leu Val Val 165 170 175 Ala Leu Gly Tyr Cys Asn AspLys Lys Asn Pro Gln Lys Asn Arg Phe 180 185 190 Ser Phe Asp Glu Val ValLys Phe Ile 195 200 24 522 DNA Porphyromonas gingivalis CDS (1)..(522)24 atg aaa aaa acg ctc gta ata gtc gtt cac ccc gat ttg acc aaa tcc 48Met Lys Lys Thr Leu Val Ile Val Val His Pro Asp Leu Thr Lys Ser 1 5 1015 gtt atc aac aag gct tgg gcc aaa gcc atc gaa ggt gca gcc act atc 96Val Ile Asn Lys Ala Trp Ala Lys Ala Ile Glu Gly Ala Ala Thr Ile 20 25 30cac cat ctc tac gaa cag tat ccg aac gga caa atc gat cta gca cat 144 HisHis Leu Tyr Glu Gln Tyr Pro Asn Gly Gln Ile Asp Leu Ala His 35 40 45 gaacaa gcc ctg ctg gag gct cat gac cgc atc gtc ttc caa ttc ccc 192 Glu GlnAla Leu Leu Glu Ala His Asp Arg Ile Val Phe Gln Phe Pro 50 55 60 ctc tattgg tat gca gct ccc tat ctg ctg aag aag tgg atg gac gag 240 Leu Tyr TrpTyr Ala Ala Pro Tyr Leu Leu Lys Lys Trp Met Asp Glu 65 70 75 80 gtc tttact gag ggc tgg gcc tat ggt gcc ggt gga gac aag atg gag 288 Val Phe ThrGlu Gly Trp Ala Tyr Gly Ala Gly Gly Asp Lys Met Glu 85 90 95 ggt aaa gaaatc tgt gca gca gtc tcc tgc gga tca ccc aaa tca gct 336 Gly Lys Glu IleCys Ala Ala Val Ser Cys Gly Ser Pro Lys Ser Ala 100 105 110 ttt gcc gaagga gca cag caa tgc cac acg ctg cga agc tac ttg aat 384 Phe Ala Glu GlyAla Gln Gln Cys His Thr Leu Arg Ser Tyr Leu Asn 115 120 125 gta ttc gacggg ata gct gct ttc ctg cgc gct cga ttc acc ggc tac 432 Val Phe Asp GlyIle Ala Ala Phe Leu Arg Ala Arg Phe Thr Gly Tyr 130 135 140 cat gcc tgctac gat tcc tac aat cct cgc ctg ccg gaa atg ctg ccg 480 His Ala Cys TyrAsp Ser Tyr Asn Pro Arg Leu Pro Glu Met Leu Pro 145 150 155 160 gcc aactgc gaa gcc tat ctc cgc ttt atc aaa gga gaa tga 522 Ala Asn Cys Glu AlaTyr Leu Arg Phe Ile Lys Gly Glu 165 170 25 173 PRT Porphyromonasgingivalis 25 Met Lys Lys Thr Leu Val Ile Val Val His Pro Asp Leu ThrLys Ser 1 5 10 15 Val Ile Asn Lys Ala Trp Ala Lys Ala Ile Glu Gly AlaAla Thr Ile 20 25 30 His His Leu Tyr Glu Gln Tyr Pro Asn Gly Gln Ile AspLeu Ala His 35 40 45 Glu Gln Ala Leu Leu Glu Ala His Asp Arg Ile Val PheGln Phe Pro 50 55 60 Leu Tyr Trp Tyr Ala Ala Pro Tyr Leu Leu Lys Lys TrpMet Asp Glu 65 70 75 80 Val Phe Thr Glu Gly Trp Ala Tyr Gly Ala Gly GlyAsp Lys Met Glu 85 90 95 Gly Lys Glu Ile Cys Ala Ala Val Ser Cys Gly SerPro Lys Ser Ala 100 105 110 Phe Ala Glu Gly Ala Gln Gln Cys His Thr LeuArg Ser Tyr Leu Asn 115 120 125 Val Phe Asp Gly Ile Ala Ala Phe Leu ArgAla Arg Phe Thr Gly Tyr 130 135 140 His Ala Cys Tyr Asp Ser Tyr Asn ProArg Leu Pro Glu Met Leu Pro 145 150 155 160 Ala Asn Cys Glu Ala Tyr LeuArg Phe Ile Lys Gly Glu 165 170 26 552 DNA Yersinia pestis CDS(1)..(552) 26 atg atg ttg cag ccg ccg aag gtt ttg ctg ctg tat gcc catccg gaa 48 Met Met Leu Gln Pro Pro Lys Val Leu Leu Leu Tyr Ala His ProGlu 1 5 10 15 tca cag gac tcg gtc gct aac cgg gtt tta ctg caa ccg gtacag cag 96 Ser Gln Asp Ser Val Ala Asn Arg Val Leu Leu Gln Pro Val GlnGln 20 25 30 tta gaa cat gtc act gtg cac gat ctt tat gca cat tat ccg gatttc 144 Leu Glu His Val Thr Val His Asp Leu Tyr Ala His Tyr Pro Asp Phe35 40 45 ttt att gat att cat cat gag cag caa ttg cta cgt gat cat caa gtt192 Phe Ile Asp Ile His His Glu Gln Gln Leu Leu Arg Asp His Gln Val 5055 60 att gta ttt caa cat cct tta tat act tac agt tgc cct gca tta ctg240 Ile Val Phe Gln His Pro Leu Tyr Thr Tyr Ser Cys Pro Ala Leu Leu 6570 75 80 aaa gag tgg ttg gat cgg gta ctg gca cgt ggt ttc gcc aat ggc gtt288 Lys Glu Trp Leu Asp Arg Val Leu Ala Arg Gly Phe Ala Asn Gly Val 8590 95 ggc ggc cat gca ctg acg gga aag cac tgg cgc tcg gtg att acc acc336 Gly Gly His Ala Leu Thr Gly Lys His Trp Arg Ser Val Ile Thr Thr 100105 110 ggt gag cag gag gga act tac cgt att ggg gga tat aac cgt tac cca384 Gly Glu Gln Glu Gly Thr Tyr Arg Ile Gly Gly Tyr Asn Arg Tyr Pro 115120 125 atg gaa gac att ctg cgt cct ttc gaa ttg acg gcg gct atg tgc cat432 Met Glu Asp Ile Leu Arg Pro Phe Glu Leu Thr Ala Ala Met Cys His 130135 140 atg cat tgg att aat ccg atg att att tac tgg gcc aga cgc caa aag480 Met His Trp Ile Asn Pro Met Ile Ile Tyr Trp Ala Arg Arg Gln Lys 145150 155 160 ccg gaa aca ctc gcc agt cac gca caa gct tat gtg caa tgg ctgcag 528 Pro Glu Thr Leu Ala Ser His Ala Gln Ala Tyr Val Gln Trp Leu Gln165 170 175 tca ccg ctc acg aga gga ctc tga 552 Ser Pro Leu Thr Arg GlyLeu 180 27 183 PRT Yersinia pestis 27 Met Met Leu Gln Pro Pro Lys ValLeu Leu Leu Tyr Ala His Pro Glu 1 5 10 15 Ser Gln Asp Ser Val Ala AsnArg Val Leu Leu Gln Pro Val Gln Gln 20 25 30 Leu Glu His Val Thr Val HisAsp Leu Tyr Ala His Tyr Pro Asp Phe 35 40 45 Phe Ile Asp Ile His His GluGln Gln Leu Leu Arg Asp His Gln Val 50 55 60 Ile Val Phe Gln His Pro LeuTyr Thr Tyr Ser Cys Pro Ala Leu Leu 65 70 75 80 Lys Glu Trp Leu Asp ArgVal Leu Ala Arg Gly Phe Ala Asn Gly Val 85 90 95 Gly Gly His Ala Leu ThrGly Lys His Trp Arg Ser Val Ile Thr Thr 100 105 110 Gly Glu Gln Glu GlyThr Tyr Arg Ile Gly Gly Tyr Asn Arg Tyr Pro 115 120 125 Met Glu Asp IleLeu Arg Pro Phe Glu Leu Thr Ala Ala Met Cys His 130 135 140 Met His TrpIle Asn Pro Met Ile Ile Tyr Trp Ala Arg Arg Gln Lys 145 150 155 160 ProGlu Thr Leu Ala Ser His Ala Gln Ala Tyr Val Gln Trp Leu Gln 165 170 175Ser Pro Leu Thr Arg Gly Leu 180 28 633 DNA Helicobacter pylori 28atgaaatttt tggatcaaga aaaaagaaga caattgctaa acgagcgcca ttcttgcaag 60atgttcgaca gccattatga gttttctagt gaagaattag aagaaatcgc tgaaatcgct 120aggctatcgc caagctctta caacacgcag ccatggcatt ttgtgatggt tactaataag 180gatttaaaaa aacaaattgc agcgcacagc tattttaatg aagaaatgat taaaagcgct 240tcagcgttaa tggtggtatg ctctttaaaa cccagcgagt tgttacccac tggccactac 300atgcaaaacc tttacccgga gtcttataag gttagagtga tcccctcttt tgctcaaatg 360cttggcgtga gattcaacca cagcatgcaa aaattagaaa gctatatttt ggagcaatgc 420tatatcgctg tggggcaaat ttgcatgggc gtgagcttaa tgggattgga tagttgcatt 480attggaggct ttgatccttt aaaagtgggc gaagttttag aagagcgtat caataaacct 540aaaatcgcat gcttgatcgc tttgggcaag agggtggcag aagcgagcca aaaatcaaga 600aaatcaaaag ttgatgccat tacttggttg tga 633 29 210 PRT Helicobacter pylori29 Met Lys Phe Leu Asp Gln Glu Lys Arg Arg Gln Leu Leu Asn Glu Arg 1 510 15 His Ser Cys Lys Met Phe Asp Ser His Tyr Glu Phe Ser Ser Glu Glu 2025 30 Leu Glu Glu Ile Ala Glu Ile Ala Arg Leu Ser Pro Ser Ser Tyr Asn 3540 45 Thr Gln Pro Trp His Phe Val Met Val Thr Asn Lys Asp Leu Lys Lys 5055 60 Gln Ile Ala Ala His Ser Tyr Phe Asn Glu Glu Met Ile Lys Ser Ala 6570 75 80 Ser Ala Leu Met Val Val Cys Ser Leu Lys Pro Ser Glu Leu Leu Pro85 90 95 Thr Gly His Tyr Met Gln Asn Leu Tyr Pro Glu Ser Tyr Lys Val Arg100 105 110 Val Ile Pro Ser Phe Ala Gln Met Leu Gly Val Arg Phe Asn HisSer 115 120 125 Met Gln Lys Leu Glu Ser Tyr Ile Leu Glu Gln Cys Tyr IleAla Val 130 135 140 Gly Gln Ile Cys Met Gly Val Ser Leu Met Gly Leu AspSer Cys Ile 145 150 155 160 Ile Gly Gly Phe Asp Pro Leu Lys Val Gly GluVal Leu Glu Glu Arg 165 170 175 Ile Asn Lys Pro Lys Ile Ala Cys Leu IleAla Leu Gly Lys Arg Val 180 185 190 Ala Glu Ala Ser Gln Lys Ser Arg LysSer Lys Val Asp Ala Ile Thr 195 200 205 Trp Leu 210

1. A nucleic acid comprising (a) a DNA encoding a nitroreductase whichpreferentially reduces CB1954 to a cytotoxic 4-hydroxylamine (4HX)derivative instead of a non-cytotoxic 2-hydroxylamine derivative,operatively coupled to (b) a promoter for expression of the DNA, whereinthe nucleic acid is selected from the group consisting of SEQ ID NO:s10, 12,17, 20, 21, 23, 25 and
 29. 2. A viral vector, comprising:— (a) aDNA encoding a nitroreductase which preferentially reduces CB1 954 to acytotoxic 4-hydroxylamine (4HX) derivative instead of a non-cytotoxic2-hydroxylamine derivative, operatively coupled to (b) a promoter forexpression of the DNA, wherein the nucleic acid is selected from thegroup consisting of SEQ ID NO:s 10, 12, 17, 20, 21,23, 25 and
 29. 3. Amethod of preparing a nitroreductase, comprising expressing a gene in abacterial cell, wherein the gene comprises a nucleic acid selected fromthe group consisting of SEQ ID NO:s 10, 12, 17, 20, 21,23, 25 and
 29. 4.A nucleic acid comprising— (a) a DNA encoding a nitroreductase whichpreferentially reduces CB1954 to a cytotoxic 4-hydroxylamine (4HX)derivative instead of a non-cytotoxic 2-hydroxylamine derivative,operatively coupled to (b) a promoter for expression of the DNA.
 5. Aviral vector, comprising:— (a) a DNA encoding a nitroreductase whichpreferentially reduces CB1954 to a cytotoxic 4-hydroxylamine (4HX)derivative instead of a non-cytotoxic 2-hydroxylamine derivative,operatively coupled to (b) a promoter for expression of the DNA.
 6. Amethod of preparing a nitroreductase, comprising expressing a gene in abacterial cell, wherein the gene encodes a nitroreductase whichpreferentially reduces CB1954 to a cytotoxic 4-hydroxylamine (4HX)derivative instead of a non-cytotoxic 2-hydroxylamine derivative.
 7. Themethod of claim 3, further comprising combining the nitroreductase witha pharmaceutically acceptable carrier.
 8. The method of claim 6, furthercomprising combining the nitroreductase with a pharmaceuticallyacceptable carrier.